A Schematics of used system . MEFs , in which HSF1 on chromosome 8 was deleted , were stably transfected with mTag - BFP expressed under control of HSPA6 promoter subsequently transiently transfected with plasmids that expressed HSF1wt or mutant variants in an artificial operon with hrGFP , which is translated from an IRES element . B Exemplary flow cytometry data plotting BFP versus GFP , indicating gates used for . Numbers indicate fraction ( % ) of ( forward scatter singlets ) in each gate . Color indicates density from blue ( low ) to red ( high ) .

O O O O O O B-CELL_LINE O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Schematics of the used system. MEFs, in which HSF1 on chromosome 8 was deleted, were stably transfected with mTag-BFP expressed under the control of the HSPA6 promoter and subsequently transiently transfected with plasmids that expressed HSF1wt or mutant variants in an artificial operon with hrGFP, which is translated from an IRES element. B Exemplary flow cytometry data plotting BFP fluorescence versus GFP fluorescence, indicating the gates used for analysis. Numbers indicate the fraction (%) of cells (forward scatter singlets) in each gate. Color indicates the cell density from dark blue (low) to red (high).


C - H Geometric mean of BFP ( C , E ) GFP ( D , F , H ) of grown at 37 ° C ( non - stress , NS ) or incubated for 1 h at 43 ° C with a recovery period of 3 h at 37 ° C ( heat shocked , HS ) in GFPhigh ( C , D ) , GFPlow ( E , F ) , GFPlow - BFPhigh ( H ) gates . ( G ) Geometric mean of BFP weighted by relative number of in GFPlow - BFPhigh gate ( * fraction forward scatter singlets ) . Note that GFPlow gate also comprises GFPlow - BFPhigh gate . Hsf1 - 202SSS , Hsf1 - I202S , L203S , V205S ; Hsf1 - 209SSS , Hsf1 - I209S , L211S , L213S ; Hsf1 - 427SSSS , Hsf1 - M427S , L429S , L432S , L436S ; Hsf1 - 465SSS , Hsf1 - L465S , V466S , Y468S ; Hsf1 - 473SSSS , Hsf1 - L473S , F474S , L475S ,

O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O

C-H Geometric mean of BFP (C, E) and GFP fluorescence (D, F, H) of cells grown at 37°C (non-stress, NS) or incubated for 1 h at 43°C with a recovery period of 3 h at 37°C (heat shocked, HS) in the GFPhigh (C, D), GFPlow (E, F), and GFPlow-BFPhigh (H) gates. (G) Geometric mean of BFP fluorescence weighted by the relative number of cells in the GFPlow-BFPhigh gate (fluorescence * fraction forward scatter singlets). Note that the GFPlow gate also comprises the GFPlow-BFPhigh gate. Hsf1-202SSS, Hsf1-I202S,L203S,V205S; Hsf1-209SSS, Hsf1-I209S,L211S,L213S; Hsf1-427SSSS, Hsf1-M427S,L429S,L432S,L436S; Hsf1-465SSS, Hsf1-L465S,V466S,Y468S; Hsf1-473SSSS, Hsf1-L473S,F474S,L475S,L476S


A Schematics of used system . HeLa were stably transfected with mTag - BFP expressed under control of HSPA6 promoter subsequently transiently transfected with plasmids that expressed HSF1wt or mutant variants in an artificial operon with hrGFP , which is translated from an IRES element . Exemplary flow cytometry data plotting BFP versus GFP , indicating gates used for . Numbers indicate fraction ( % ) of ( forward scatter singlets ) in each gate . Color indicates density from blue ( low ) to red ( high ) . NS , grown at 37 ° C ; HS , heat shocked at 43 ° C for 1 h with a recovery period of 3 h at 37 ° C .

O O O O O O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Schematics of the used system. HeLa cells were stably transfected with mTag-BFP expressed under the control of the HSPA6 promoter and subsequently transiently transfected with plasmids that expressed HSF1wt or mutant variants in an artificial operon with hrGFP, which is translated from an IRES element. Exemplary flow cytometry data plotting BFP fluorescence versus GFP fluorescence, indicating the gates used for analysis. Numbers indicate the fraction (%) of cells (forward scatter singlets) in each gate. Color indicates the cell density from dark blue (low) to red (high). NS, cells grown at 37°C; HS, cells heat shocked at 43°C for 1 h with a recovery period of 3 h at 37°C.


C - H Geometric mean of BFP ( C , E ) GFP ( D , F , H ) in GFPhigh ( C , D ) , GFPlow ( E , F ) , GFPlow - BFPhigh43 ( H ) gates . Of note , number of data points show number of experiments in which a significant number of was found in this gate missing bars indicate that did not fall within this gate in more than two out of four experiments . ( G ) Geometric mean of BFP weighted by relative number of in GFPlow - BFPhigh43 gate ( * fraction forward scatter singlets ) . Note that GFPlow gate also comprises GFPlow - BFPhigh37 that GFPlow - BFPhigh37 gate also comprises GFPlow - BFPhigh43 gate . Hsf1 - 202SSS , Hsf1 - I202S , L203S , V205S ; Hsf1 - 209SSS , Hsf1 - I209S , L211S , L213S ; Hsf1 - 427SSSS , Hsf1 - M427S , L429S , L432S , L436S ; Hsf1 - 465SSS , Hsf1 - L465S , V466S , Y468S ; Hsf1 - 473SSSS , Hsf1 - L473S , F474S , L475S ,

O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O

C-H Geometric mean of BFP (C, E) and GFP fluorescence (D, F, H) in the GFPhigh (C, D), GFPlow (E, F), and GFPlow-BFPhigh43 (H) gates. Of note, the number of data points show the number of experiments in which a significant number of cells was found in this gate and missing bars indicate that cells did not fall within this gate in more than two out of four experiments. (G) Geometric mean of BFP fluorescence weighted by the relative number of cells in the GFPlow-BFPhigh43 gate (fluorescence * fraction forward scatter singlets). Note that the GFPlow gate also comprises the GFPlow-BFPhigh37 and that the GFPlow-BFPhigh37 gate also comprises the GFPlow-BFPhigh43 gate. Hsf1-202SSS, Hsf1-I202S,L203S,V205S; Hsf1-209SSS, Hsf1-I209S,L211S,L213S; Hsf1-427SSSS, Hsf1-M427S,L429S,L432S,L436S; Hsf1-465SSS, Hsf1-L465S,V466S,Y468S; Hsf1-473SSSS, Hsf1-L473S,F474S,L475S,L476S


At 72 h after transfection with siRNA duplexes for BAG6 or control siRNA ( 10 nM each ) , intracellular localization of TfnR in HeLa was examined ( shown as green ) . was stained with Hoechst 33342 ( shown as blue ) . Control knockdown ( left panel ) , Rab8a knockdown ( center panel ) , BAG6 knockdown ( right panel ) .

O O O O O O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-CELL_LINE O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O

At 72 h after transfection with siRNA duplexes for BAG6 or control siRNA (10 nM each), the intracellular localization of TfnR in HeLa cells was examined (shown as green). Nuclear DNA was stained with Hoechst 33342 (shown as blue). Control knockdown (left panel), Rab8a knockdown (center panel), and BAG6 knockdown (right panel).


Intracellular localization of Ptc1 ( green ) in HeLa . Nuclei were stained with Hoechst 33342 ( shown as blue ) .

O B-EXP_ASSAY O B-GENEPROD O O O O B-CELL_LINE O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O

Intracellular localization of Ptc1 (green) in HeLa cells. Nuclei were stained with Hoechst 33342 (shown as blue).


Knockdown of Rab8a ( with Rab8a siRNA # 1 , # 2 , # 3 ) stimulated accumulation stabilization of Ptc1 in HEK293 .

O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O B-CELL_LINE O

Knockdown of Rab8a (with Rab8a siRNA#1, #2, and #3) stimulated the accumulation and stabilization of Ptc1 protein in HEK293 cells.


Knockdown of BAG6 ( with BAG6 siRNA # 1 ) stimulated accumulation stabilization of Ptc1 in HEK293 .

O O B-GENEPROD O O B-GENEPROD O O O O O O O O B-GENEPROD O B-CELL_LINE O

Knockdown of BAG6 (with BAG6 siRNA#1) stimulated the accumulation and stabilization of Ptc1 protein in HEK293 cells.


BAG6 co - precipitated Rab8a , while neither Rab7 nor Rab11a were co - precipitated with BAG6 . Flag - tagged Rab8a , Rab7 , Rab11a , luciferase - CL1 ( Lc - CL1 ; a positive control ) were expressed in HeLa were treated with 10 μM MG - 132 for 4 h . Flag - immunoprecipitates were blotted with anti - BAG6 anti - Flag antibodies , respectively . Note that used were treated with 10 μM MG - 132 for 4 h .

B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-CELL_LINE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD O B-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O

BAG6 protein co-precipitated Rab8a, while neither Rab7 nor Rab11a were co-precipitated with BAG6. Flag-tagged Rab8a, Rab7, Rab11a, and luciferase-CL1 (Lc-CL1; a positive control) were expressed in HeLa cells and the cells were treated with 10 μM MG-132 for 4 h. Flag-immunoprecipitates were blotted with anti-BAG6 and anti-Flag antibodies, respectively. Note that all cells used were treated with 10 μM MG-132 for 4 h.


S - tagged BAG6 pull - down efficiently co - precipitated Rab8a ( T22N ) , a GDP - bound mutant , while BAG6 scarcely co - precipitated Rab8a ( Q67L ) , a constitutively active mutant . Co - precipitation of Rab8a WT with BAG6 was used as a standard . MG - 132 ( 10 μM ) was included in culture for 4 h , as indicated . Note that T22N mutant was expressed at lower than either WT or Q67L , that this was partly due to increased degradation , as will be shown later . S - BAG6 stands for N - terminally S - tagged BAG6

B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD

S-tagged BAG6 pull-down efficiently co-precipitated Rab8a (T22N), a GDP-bound mutant, while BAG6 scarcely co-precipitated Rab8a (Q67L), a constitutively active mutant. Co-precipitation of Rab8a WT with BAG6 was used as a standard. MG-132 (10 μM) was included in the cell culture for 4 h, as indicated. Note that the T22N mutant protein was expressed at lower levels than either WT or Q67L, and that this was partly due to increased degradation, as will be shown later. S-BAG6 stands for N-terminally S-tagged BAG6 protein


Anti - Flag signals in ( C ) were quantified , relative signal intensities are presented . value of WT Rab8a signal with MG - 132 was defined as 1 . 0 . Note that signal intensities of Flag - tag were normalized by that of Rab8a input signals .

O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O B-GENEPROD O O O

Anti-Flag signals in (C) were quantified, and relative signal intensities are presented. The value of the WT Rab8a signal with MG-132 was defined as 1.0. Note that all signal intensities of Flag-tag were normalized by that of the Rab8a input signals.


A series of Flag - Rab8a mutants were immunoprecipitated quantified amount of endogenous BAG6 that were coprecipitated with Flag - Rab8a . Note that used were treated with 10 μM MG - 132 for 4 h .

O O O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O

A series of Flag-Rab8a mutants were immunoprecipitated and quantified the amount of endogenous BAG6 that were coprecipitated with Flag-Rab8a. Note that all cells used were treated with 10 μM MG-132 for 4 h.


Deficiency of Rabin8 , a GEF for Rab8a , enhanced physical interaction between BAG6 WT Rab8a . Flag - tagged WT Rab8a was expressed in Rabin8 siRNA - treated , Flag immunoprecipitates were probed with an anti - BAG6 antibody . Note that used were treated with 10 μM MG - 132 for 4 h .

O O B-GENEPROD O O O O B-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O

Deficiency of Rabin8, a GEF for Rab8a, enhanced the physical interaction between BAG6 and WT Rab8a proteins. Flag-tagged WT Rab8a was expressed in Rabin8 siRNA-treated cells, and Flag immunoprecipitates were probed with an anti-BAG6 antibody. Note that all cells used were treated with 10 μM MG-132 for 4 h.


C - terminal geranylgeranylation of Rab8a was dispensable for BAG6 recognition .

O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O

C-terminal geranylgeranylation of Rab8a was dispensable for BAG6 recognition.


N - terminal GTPase domain of Rab8a was essential for BAG6 recognition . A series of Flag - tagged truncated fragments of WT Rab8a were expressed in HeLa with S - tagged BAG6 treated with ( + ) or without ( - ) protease inhibitors for 4 h . Flag - Rab8a substrates were immunoprecipitated probed with an anti - BAG6 antibody .

O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-CELL_LINE O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O O O O O B-GENEPROD O O

The N-terminal GTPase domain of Rab8a was essential for BAG6 recognition. A series of Flag-tagged truncated fragments of WT Rab8a were expressed in HeLa cells with S-tagged BAG6 and treated with (+) or without (-) protease inhibitors for 4 h. Flag-Rab8a substrates were immunoprecipitated and probed with an anti-BAG6 antibody.


Hydrophobicity recognition domain of BAG6 ( N465 ) was critical for Rab8a recognition . A schematic representation of BAG6 - truncated used in this experiment is shown in upper panel . Numbers denote corresponding of mammalian BAG6 . Positions of UBL , BUILD , DUF3538 domains , which are linked to hydrophobicity recognition by BAG6 [ 37 ] , are indicated . WT , wild - type ; ∆ N465 , N - terminal 465 residues - deleted mutant ; Ν465 , C - terminal 689 residues - deleted mutant .

O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Hydrophobicity recognition domain of BAG6 (N465) was critical for Rab8a recognition. A schematic representation of the BAG6-truncated proteins used in this experiment is shown in the upper panel. Numbers denote the corresponding amino acids of mammalian BAG6. Positions of UBL, BUILD, and DUF3538 domains, which are all linked to hydrophobicity recognition by BAG6 [37], are indicated. WT, wild-type; ∆N465, N-terminal 465 residues-deleted mutant; and Ν465, C-terminal 689 residues-deleted mutant.


CHX chase experiments show that GDP - bound Rab8a ( T22N ) was a highly labile , while GTP - bound active mutant ( Q67L ) was a stable ( E ) . Instability of T22N mutant was not perturbed by C204S mutation ( F ) . Actin was used as a loading control .

B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O

CHX chase experiments show that GDP-bound Rab8a (T22N) was a highly labile protein, while the GTP-bound active mutant (Q67L) was a stable protein (E). Instability of the T22N mutant was not perturbed by the C204S mutation (F). Actin was used as a loading control.


N - terminal 100 residue fragment of Rab8a GTPase domain was sensitive to proteasome inhibitor , while ∆ N100 fragment was not .

O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O

N-terminal 100 residue fragment of the Rab8a GTPase domain was sensitive to the proteasome inhibitor, while the ∆N100 fragment was not.


Rab8a ( T22N ) was polyubiquitinated in presence of proteasome inhibitor . Asterisks indicate non - specific bands .

B-GENEPROD O O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O

Rab8a (T22N) was polyubiquitinated in the presence of the proteasome inhibitor. Asterisks indicate non-specific bands.


Kyte - Doolittle hydrophobicity plots of complete acid of human WT Rab8a T22N - 3IS mutant . hydrophobicity peak within WT Switch I was abolished in 3IS mutant ( indicated within red box ) . numbers on horizontal axis denote corresponding acid positions in these .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O B-ORGANISM O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O

Kyte-Doolittle hydrophobicity plots of the complete amino acid sequence of human WT Rab8a and T22N-3IS mutant. The hydrophobicity peak within WT Switch I was abolished in the 3IS mutant (indicated within the red box). The numbers on the horizontal axis denote the corresponding amino acid positions in these proteins.


Substitution of hydrophobic residues of Switch I with hydrophilic residues ( T22N 3IS ) abolished its binding to BAG6 .

O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O

Substitution of the hydrophobic residues of Switch I with hydrophilic residues (T22N 3IS) abolished its binding to BAG6 protein.


Rab8a ( T22N - 3IS ) mutant was highly stable , while Rab8a ( T22N ) was quite unstable in HeLa . Actin was used as a loading control .

B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O B-CELL_LINE O B-GENEPROD O O O O O O O

Rab8a (T22N-3IS) mutant protein was highly stable, while Rab8a (T22N) protein was quite unstable in HeLa cells. Actin was used as a loading control.


Rab8a ( T22N - 3IS ) mutant was not subject to polyubiquitin co - precipitation , while Rab8a ( T22N ) was .

B-GENEPROD O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O

Rab8a (T22N-3IS) mutant was not subject to polyubiquitin co-precipitation, while Rab8a (T22N) protein was.


Rab8a ( T22N ) accumulated in BAG6 - knockdown . HeLa were transfected with siRNA duplexes for BAG6 or control siRNA . At 48 h after siRNA transfection , Flag tagged - Rab8a ( T22N ) was expressed in . At 24 h after Rab8a ( T22N ) transfection , were chased with 50 μg / mL CHX harvested at indicated time after CHX addition . Actin was used as a loading control .

B-GENEPROD O O O O O B-GENEPROD O O O B-CELL_LINE O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O

Rab8a (T22N) protein accumulated in BAG6-knockdown cells. HeLa cells were transfected with siRNA duplexes for BAG6 or control siRNA. At 48 h after siRNA transfection, Flag tagged-Rab8a (T22N) was expressed in the cells. At 24 h after Rab8a (T22N) transfection, the cells were chased with 50 μg/mL CHX and harvested at the indicated time after CHX addition. Actin was used as a loading control.


Anti - Flag blot signals in control or BAG6 siRNA - treated were quantified , relative signal intensities after CHX addition were calculated . value of Flag signal at 0 h was defined as 1 . 0 . Note that signal intensities of Flag - tag were normalized by that of actin , a loading control , in each sample .

O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O

Anti-Flag blot signals in the control or BAG6 siRNA-treated cells were quantified, and relative signal intensities after CHX addition were calculated. The value of the Flag signal at 0 h was defined as 1.0. Note that all signal intensities of the Flag-tag were normalized by that of actin, a loading control, in each sample.


Polyubiquitin modification of Rab8a was abolished in BAG6 - knockdown . Flag - Rab8a ( T22N ) immunoprecipitates were blotted with an anti - polyubiquitin antibody ( FK2 , left panel ) . As a negative control , siRNA # 1scr was used . Anti - polyubiquitin signals co - precipitated with Rab8a ( T22N ) ( a representative example is shown in left panel ) were quantified ( right panel ) . Note that intensities of co - precipitated polyubiquitin signal were normalized both by input ubiquitin - signal bait Flag - signal .

B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O O O

Polyubiquitin modification of Rab8a was abolished in BAG6-knockdown cells. Flag-Rab8a (T22N) immunoprecipitates were blotted with an anti-polyubiquitin antibody (FK2, left panel). As a negative control, siRNA#1scr was used. Anti-polyubiquitin signals co-precipitated with Rab8a (T22N) (a representative example is shown in the left panel) were quantified (right panel). Note that the intensities of the co-precipitated polyubiquitin signal were normalized both by the input ubiquitin-signal and bait Flag-signal.


Defective distribution of endosomal TfnR in HeLa with excess accumulation of inactive form of Rab8a . Right panel shows a merged image of TfnR ( shown as green ) , Rab8a ( T22N ) ( magenta ) , Hoechst 33342 ( blue ) .

O O O B-SUBCELLULAR B-GENEPROD O B-CELL_LINE O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O

Defective distribution of the endosomal protein TfnR in HeLa cells with the excess accumulation of the inactive form of Rab8a. Right panel shows a merged image of TfnR staining (shown as green), Rab8a (T22N) staining (magenta), and Hoechst 33342 nuclear staining (blue).


A series of major Rab family were immunoprecipitated from HeLa extracts then probed with an anti - BAG6 antibody . Co - precipitation of Rab8a ( T22N ) with BAG6 was used as a positive control for this experiment .

O O O O O O O B-EXP_ASSAY O B-CELL_LINE O O O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O

A series of major Rab family proteins were immunoprecipitated from HeLa cell extracts and then probed with an anti-BAG6 antibody. Co-precipitation of Rab8a (T22N) with BAG6 was used as a positive control for this experiment.


of BAG6 co - precipitation with Rab family . graph shows quantities of BAG6 in respective anti - Flag precipitates that were normalized by amount of Flag - Rab bait . value of BAG6 co - precipitation with Rab8a ( T22N ) was defined as 1 . 0 . quantities of respective immunosignals were also normalized to actin signal of each sample , represent median calculated from at least 3 independent biological replicates . number of independent biological replicates was as follows : Rab1a , n = 4 ; Rab1b , n = 5 ; Rab2b , n = 3 ; Rab3a , n = 4 ; Rab4 , n = 4 ; Rab5a , n = 3 ; Rab5b , n = 4 ; Rab6a , n = 5 ; Rab6b , n = 4 ; Rab7a , n = 3 ; Rab8a , n = 14 ; Rab9b , n = 4 ; Rab10 , n = 6 ; Rab11a , n = 3 ; Rab11b , n = 3 ; Rab12 , n = 2 ; Rab13 , n = 6 ; Rab14 , n = 3 ; Rab15 , n = 5 ; Rab35 , n = 3 .

O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O

Quantification of BAG6 co-precipitation with Rab family proteins. The graph shows the quantities of BAG6 protein in the respective anti-Flag precipitates that were normalized by the amount of Flag-Rab bait proteins. The value of BAG6 co-precipitation with Rab8a (T22N) was defined as 1.0. The quantities of the respective immunosignals were also normalized to the actin signal of each sample, and represent the median calculated from at least 3 independent biological replicates. The number of independent biological replicates was as follows: Rab1a, n = 4; Rab1b, n = 5; Rab2b, n = 3; Rab3a, n = 4; Rab4, n = 4; Rab5a, n = 3; Rab5b, n = 4; Rab6a, n = 5; Rab6b, n = 4; Rab7a, n = 3; Rab8a, n = 14; Rab9b, n = 4; Rab10, n = 6; Rab11a, n = 3; Rab11b, n = 3; Rab12, n = 2; Rab13, n = 6; Rab14, n = 3; Rab15, n = 5; Rab35, n = 3.


BAG6 knockdown induced abnormal distribution of Golgi apparatus markers . Representative of trans - Golgi Stx6 ( green ) in BAG6 - suppressed CHO with a Chinese hamster - specific siRNA ( cBAG6 siRNA # 2 ) . Scale bar : 10 μm ( A ) . signals were detected using a scanning confocal microscopy system . Nuclei were stained by Hoechst ( blue ) .

B-GENEPROD O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-SUBCELLULAR B-GENEPROD O O O O B-GENEPROD O O B-CELL_LINE O O B-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O

BAG6 knockdown induced the abnormal distribution of Golgi apparatus markers. Representative images of the trans-Golgi membrane protein Stx6 (green) in BAG6-suppressed CHO cells with a Chinese hamster-specific siRNA (cBAG6 siRNA#2). Scale bar: 10 μm (A). Fluorescent signals were detected using a laser scanning confocal microscopy system. Nuclei were stained by Hoechst (blue).


BAG6 knockdown induced abnormal distribution of Golgi apparatus markers . of cis - Golgi GS28 cis - Golgi matrix GM130 in BAG6 - suppressed CHO with another Chinese hamster - specific siRNA ( cBAG6 siRNA # 5 ) . GS28 ( green ) GM130 ( red ) are indicated by arrowheads . Scale bar : 10 μm . ( B ) . signals were detected using a scanning confocal microscopy system . Nuclei were stained by Hoechst ( blue ) .

B-GENEPROD O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O B-ORGANISM I-ORGANISM O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O

BAG6 knockdown induced the abnormal distribution of Golgi apparatus markers. Images of the cis-Golgi membrane protein GS28 and the cis-Golgi matrix protein GM130 in BAG6-suppressed CHO cells with another Chinese hamster-specific siRNA (cBAG6 siRNA#5). GS28 (green) and GM130 (red) are indicated by arrowheads. Scale bar: 10 μm. (B). Fluorescent signals were detected using a laser scanning confocal microscopy system. Nuclei were stained by Hoechst (blue).


Glycosylation of IL - 2Rα transmembrane was not reduced by BAG6 knockdown . Flag - tagged WT IL - 2Rα was expressed in HeLa with ( + ) or without ( - ) BAG6 siRNA , was immunoprecipitated with an anti - Flag antibody . precipitates were incubated with ( + ) or without ( - ) 10 unit of deglycosylation enzyme PNGase F , subjected to Western blot with an anti - Flag antibody . Low - mobility ( indicated as glycosylated ) high mobility ( indicated as non - glycosylated ) signals of WT IL - 2Rα are indicated .

B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_LINE O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O

Glycosylation of the IL-2Rα transmembrane protein was not reduced by BAG6 knockdown. Flag-tagged WT IL-2Rα protein was expressed in HeLa cells with (+) or without (-) BAG6 siRNA, and was immunoprecipitated with an anti-Flag antibody. The precipitates were incubated with (+) or without (-) 10 unit of the deglycosylation enzyme PNGase F, and subjected to Western blot analysis with an anti-Flag antibody. Low-mobility (indicated as glycosylated) and high mobility (indicated as non-glycosylated) signals of WT IL-2Rα are indicated.


Defects in distribution of surface glycoproteins in BAG6 - suppressed . graph quantitatively displays number of per as mean ± S . D . calculated from 10 independent biological replicates ( E ) .

O O O O B-SUBCELLULAR B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O

Defects in the distribution of cell surface glycoproteins in BAG6-suppressed cells. The graph quantitatively displays the number of fluorescence counts per cell as the mean ± S.D. calculated from 10 independent biological replicates (E).


Defects in distribution of surface glycoproteins in BAG6 - suppressed . Lectin GS - II - derived surface signals were counted using ImageJ software . SRP54 knockdown was used as a positive control for this experiment t - ) . surface signals were detected by confocal microscopy without plasma permeabilization . BAG6 siRNA down - regulated surface expression of glycoproteins ( F ) .

O O O O B-SUBCELLULAR B-SMALL_MOLECULE O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-SUBCELLULAR O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O B-GENEPROD O O O O B-SUBCELLULAR B-EXP_ASSAY O B-SMALL_MOLECULE O O O O

Defects in the distribution of cell surface glycoproteins in BAG6-suppressed cells. Lectin GS-II-derived cell surface signals were counted using ImageJ software. SRP54 knockdown was used as a positive control for this experiment t-test). Cell surface fluorescent signals were detected by confocal microscopy without plasma membrane permeabilization. BAG6 siRNA down-regulated the cell surface expression of glycoproteins (F).


( a ) Densitometric relative to actin of A53T α - synuclein clearance in stable inducible PC12 expressing A53T α - synuclein . Transgene expression was induced with doxycycline for 48 h , then switched off ( by removing doxycycline ) , with drug ( 1 μM ) or DMSO ( control ) treatment for 24 h . Control condition is set to 100 % . Error bars show s . e . m .

O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O

(a) Densitometric analysis relative to actin of A53T α-synuclein clearance in stable inducible PC12 cell line expressing A53T α-synuclein. Transgene expression was induced with doxycycline for 48 h, and then switched off (by removing doxycycline), with drug (all 1 μM) or DMSO (control) treatment for 24 h. Control condition is set to 100%. Error bars show s.e.m.


( b ) Densitometric relative to actin of soluble EGFP - HDQ74 clearance in stable inducible PC12 expressing EGFP - HDQ74 . Transgene expression was induced with doxycycline for 8 h , then switched off ( by removing doxycycline ) with drug ( 1 μM ) or DMSO ( control ) treatment for 96 h . Control condition is set to 100 % . Error bars show s . e . m .

O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O B-CELL_LINE O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O

(b) Densitometric analysis relative to actin of soluble EGFP-HDQ74 clearance in stable inducible PC12 cell line expressing EGFP-HDQ74. Transgene expression was induced with doxycycline for 8 h, and then switched off (by removing doxycycline) with drug (all 1 μM) or DMSO (control) treatment for 96 h. Control condition is set to 100%. Error bars show s.e.m.


( c ) SK - N - SH transfected with EGFP - HDQ74 construct for 4 h were treated with drugs ( 1 μM ) or DMSO ( control ) for 48 h post - transfection . proportions of EGFP - positive with or were expressed as odds ratios control was taken as 1 . Error bars show 95 % confidence interval .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O

(c) SK-N-SH cells transfected with EGFP-HDQ74 construct for 4 h were treated with drugs (all 1 μM) or DMSO (control) for 48 h post-transfection. The proportions of EGFP-positive cells with aggregates or cell death were expressed as odds ratios and the control was taken as 1. Error bars show 95% confidence interval.


( d ) PC12 were treated with clonidine , minoxidil verapamil ( 1 μM ) or DMSO ( control ) for 24 h . Endogenous LC3 - II were detected with anti - LC3 antibody quantified relative to actin . Rapamycin ( 0 . 2 μM ) was positive control . Error bars show s . e . m .

O O O B-CELL_LINE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O

(d) PC12 cells were treated with clonidine, minoxidil and verapamil (all 1 μM) or DMSO (control) for 24 h. Endogenous LC3-II levels were detected with anti-LC3 antibody and quantified relative to actin. Rapamycin (0.2 μM) was positive control. Error bars show s.e.m.


( e ) proportions of EGFP - positive with EGFP - HDQ74 in wild - type ( Atg5 + / + ) knockout ( Atg5 − / − ) Atg5 MEFs , transfected with EGFP - HDQ74 for 4 h then treated for 48 h with drugs ( 1 μM ) or DMSO ( control ) . Error bars show 95 % confidence interval . * * * P 0 . 001 ; * * P 0 . 01 ; * P 0 . 05 ; NS , nonsignificant .

O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-GENEPROD B-CELL_LINE O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(e) The proportions of EGFP-positive cells with EGFP-HDQ74 aggregates in wild-type (Atg5+/+) and knockout (Atg5−/−) Atg5 MEFs, transfected with EGFP-HDQ74 for 4 h and then treated for 48 h with drugs (all 1 μM) or DMSO (control). Error bars show 95% confidence interval. ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.


( a ) A53T α - synuclein clearance in stable PC12 as in Figure 1a , treated with or without 1 μM rilmenidine , 1 mM dibutyl - cAMP ( db - cAMP ) , 24 μM forskolin or 500 μM 2 ′ 5 ′ - dideoxyadenosine ( 2 ′ 5 ′ ddA ) for 24 h . Note that control clearance for a given substrate may vary from figure to figure , due to different exposures of blots , which aid illustration of agents that either increase or retard clearance .

O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_LINE O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) A53T α-synuclein clearance in stable PC12 cells as in Figure 1a, treated with or without 1 μM rilmenidine, 1 mM dibutyl-cAMP (db-cAMP), 24 μM forskolin or 500 μM 2′5′-dideoxyadenosine (2′5′ddA) for 24 h. Note that control clearance levels for a given substrate may vary from figure to figure, due to different exposures of blots, which aid illustration of agents that either increase or retard clearance.


( b ) proportions of EGFP - positive with EGFP - HDQ74 in SK - N - SH ( for rilmenidine ) COS - 7 ( for db - cAMP , forskolin 2 ′ 5 ′ ddA ) as in Figure 1c , treated with concentrations used in a for 48 h post - transfection . Error bars show 95 % confidence interval .

O O O O O B-GENEPROD O O O B-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-SMALL_MOLECULE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O

(b) The proportions of EGFP-positive cells with EGFP-HDQ74 aggregates in SK-N-SH (for rilmenidine) and COS-7 (for db-cAMP, forskolin and 2′5′ddA) cells as in Figure 1c, treated with concentrations used in a for 48 h post-transfection. Error bars show 95% confidence interval.


( c ) Endogenous LC3 - II in PC12 treated with 1 μM rilmenidine or 500 μM 2 ′ 5 ′ ddA for 24 h .

O O O O B-GENEPROD O O O B-CELL_LINE O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O

(c) Endogenous LC3-II levels in PC12 cells treated with 1 μM rilmenidine or 500 μM 2′5′ddA for 24 h.


( d ) A53T α - synuclein clearance in stable PC12 as in Figure 1a , treated with or without 8 - CPT - 2Me - cAMP or 6 - Bnz - cAMP ( 1 μM or 10 μM ) .

O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_LINE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O

(d) A53T α-synuclein clearance in stable PC12 cells as in Figure 1a, treated with or without 8-CPT-2Me-cAMP or 6-Bnz-cAMP (1 μM or 10 μM).


( e ) proportions of EGFP - positive COS - 7 with as in Figure 1c , treated with or without 10 μM 8 - CPT - 2Me - cAMP or 6 - Bnz - cAMP for 48 h post - transfection . Error bars show 95 % confidence interval .

O O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O

(e) The proportions of EGFP-positive COS-7 cells with aggregates as in Figure 1c, treated with or without 10 μM 8-CPT-2Me-cAMP or 6-Bnz-cAMP for 48 h post-transfection. Error bars show 95% confidence interval.


( f ) COS - 7 , transfected for 4 h with EGFP - HDQ74 along with empty vector ( pcDNA3 . 1 ) , dominant - negative S17N Rap2B ( DN Rap2B ) or wild - type PLC - ε ( 1 : 3 ) , were assessed for proportions of EGFP - positive with after 48 h . Error bars show 95 % confidence interval .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O

(f) COS-7 cells, transfected for 4 h with EGFP-HDQ74 along with empty vector (pcDNA3.1), dominant-negative S17N Rap2B (DN Rap2B) or wild-type PLC-ε (1:3 ratio), were assessed for the proportions of EGFP-positive cells with aggregates after 48 h. Error bars show 95% confidence interval.


( g ) Endogenous LC3 - II in COS - 7 transfected with a dominant - negative Rap2B ( DN Rap2B ) for 4 h were assessed after 48 h expression .

O O O O B-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O

(g) Endogenous LC3-II levels in COS-7 cells transfected with a dominant-negative Rap2B (DN Rap2B) for 4 h were assessed after 48 h expression.


( h ) proportions of EGFP - positive COS - 7 with , transfected with EGFP - HDQ74 along with empty vector ( pcDNA3 . 1 ) or dominant - negative Rap2B ( 1 : 3 ) for 4 h , were treated with or without 10 μM 8 - CPT - 2Me - cAMP for 48 h . Error bars show 95 % confidence interval .

O O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O

(h) The proportions of EGFP-positive COS-7 cells with aggregates, transfected with EGFP-HDQ74 along with empty vector (pcDNA3.1) or dominant-negative Rap2B (1:3 ratio) for 4 h, were treated with or without 10 μM 8-CPT-2Me-cAMP for 48 h. Error bars show 95% confidence interval.


( i ) COS - 7 , transfected with EGFP - HDQ74 dsRed2 or dsRed2 - IP3 kinase A ( 1 : 3 ) for 4 h , were assessed at 48 h post - transfection for aggregation ( as in Fig . 1c ) . Error bars show 95 % confidence interval . * * * P 0 . 001 ; * * P 0 . 01 ; * P 0 . 05 ; NS , nonsignificant .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(i) COS-7 cells, transfected with EGFP-HDQ74 and dsRed2 or cytosolic dsRed2-IP3 kinase A (1:3 ratio) for 4 h, were assessed at 48 h post-transfection for aggregation and cell death (as in Fig. 1c). Error bars show 95% confidence interval. ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.


( a ) SK - N - SH were pretreated with or without 10 , 15 or 20 μM calpastatin for 15 min followed by addition of 1 μM ( ± ) - Bay K8644 for 4 h . Calpain activity was detected by immunoblotting with anti - calpain small subunit antibody . Densitometry is relative to actin . Error bars show s . e . m .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O

(a) SK-N-SH cells were pretreated with or without 10, 15 or 20 μM calpastatin for 15 min followed by addition of 1 μM (±)-Bay K8644 for 4 h. Calpain activity was detected by immunoblotting with anti-calpain small subunit antibody. Densitometry analysis is relative to actin. Error bars show s.e.m.


( b ) SK - N - SH transfected with EGFP - HDQ74 for 4 h , then pretreated with or without 10 , 15 or 20 μM calpastatin for 15 min followed by addition of 1 μM ( ± ) - Bay K8644 for 48 h post - transfection , were assessed for proportion of EGFP - positive with EGFP - HDQ74 . Error bars show 95 % confidence interval .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O

(b) SK-N-SH cells transfected with EGFP-HDQ74 for 4 h, then pretreated with or without 10, 15 or 20 μM calpastatin for 15 min followed by addition of 1 μM (±)-Bay K8644 for 48 h post-transfection, were assessed for the proportion of EGFP-positive cells with EGFP-HDQ74 aggregates. Error bars show 95% confidence interval.


( c ) SK - N - SH were pretreated with or without 10 , 15 or 20 μM calpastatin for 15 min followed by addition of 1 μM ( ± ) - Bay K8644 for 4 h . Endogenous LC3 - II were detected by immunoblotting with anti - LC3 antibody .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O B-GENEPROD O O

(c) SK-N-SH cells were pretreated with or without 10, 15 or 20 μM calpastatin for 15 min followed by addition of 1 μM (±)-Bay K8644 for 4 h. Endogenous LC3-II levels were detected by immunoblotting with anti-LC3 antibody.


( d ) Comparison between calpain activity , EGFP - HDQ74 aggregation autophagosomes as seen in a - c . control condition for assessments was set at 100 % . Calpastatin lowered increased calpain activity EGFP - HDQ74 aggregation caused by ( ± ) - Bay K8644 in a dose - dependent manner with a simultaneous increase in LC3 - II ( autophagosomes ) .

O O O O O B-GENEPROD O O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O O B-SUBCELLULAR O O

(d) Comparison between calpain activity, EGFP-HDQ74 aggregation and autophagosomes as seen in a-c. The control condition for all the assessments was set at 100%. Calpastatin lowered the increased calpain activity and EGFP-HDQ74 aggregation caused by (±)-Bay K8644 in a dose-dependent manner with a simultaneous increase in LC3-II levels (autophagosomes).


( e ) COS - 7 transfected with empty vector ( pcDNA3 . 1 ) or wild - type PLC - ε for 4 h treated with or without 50 μM calpeptin for 48 h post - transfection were assessed for proportion of EGFP - positive with EGFP - HDQ74 . Error bars show 95 % confidence interval . * * * P 0 . 001 ; * * P 0 . 01 ; * P 0 . 05 ; NS , nonsignificant .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(e) COS-7 cells transfected with empty vector (pcDNA3.1) or wild-type PLC-ε for 4 h and treated with or without 50 μM calpeptin for 48 h post-transfection were assessed for the proportion of EGFP-positive cells with EGFP-HDQ74 aggregates. Error bars show 95% confidence interval. ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.


( a ) proportions of EGFP - positive SK - N - SH with EGFP - HDQ74 or as in Figure 1c , treated with or without 10 μM calpastatin , 50 μM N - acetyl - Leu - Leu - methional ( ALLM ) or 50 μM calpeptin for 48 h post - transfection . DMSO was control for ALLM calpeptin . Error bars show 95 % confidence interval .

O O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O

(a) The proportions of EGFP-positive SK-N-SH cells with EGFP-HDQ74 aggregates or cell death as in Figure 1c, treated with or without 10 μM calpastatin, 50 μM N-acetyl-Leu-Leu-methional (ALLM) or 50 μM calpeptin for 48 h post-transfection. DMSO was control for ALLM and calpeptin. Error bars show 95% confidence interval.


( b ) HeLa were transfected with EGFP - HDQ74 construct along with siGLO ( control siRNA ) in presence or absence of calpain 1 or calpain 2 siRNA ( 1 : 3 ) for 96 h . EGFP - siGLO - positive were assessed for aggregation as in Figure 1c . Error bars show 95 % confidence interval .

O O O B-CELL_LINE O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

(b) HeLa cells were transfected with EGFP-HDQ74 construct along with siGLO (control siRNA) in presence or absence of calpain 1 or calpain 2 siRNA (1:3 ratio) for 96 h. EGFP- and siGLO-positive cells were assessed for aggregation and cell death as in Figure 1c. Error bars show 95% confidence interval.


( c ) HeLa , transfected with siGLO or siRNAs for calpain 1 or calpain 2 for 96 h , were analyzed with anti - calpain 1 or anti - calpain 2 domain III antibodies .

O O O B-CELL_LINE O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD O O O O

(c) HeLa cells, transfected with siGLO or siRNAs for calpain 1 or calpain 2 for 96 h, were analyzed with anti-calpain 1 or anti-calpain 2 domain III antibodies.


( d ) Clearance of soluble EGFP - HDQ74 in stable PC12 as in Figure 1b , treated with drugs ( concentrations in a ) for 96 h . Densitometry is relative to actin . Error bars show s . e . m .

O O O O O O B-GENEPROD O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O

(d) Clearance of soluble EGFP-HDQ74 in stable PC12 cells as in Figure 1b, treated with drugs (concentrations in a) for 96 h. Densitometry is relative to actin. Error bars show s.e.m.


( e ) A53T α - synuclein clearance in undifferentiated or nonmitotic neuronally differentiated ( with 100 ng ml - 1 nerve growth factor for 5 d ) stable PC12 as in Figure 1a , treated with or without 10 μM calpastatin for 24 h . Densitometry is relative to actin . Error bars show s . e . m .

O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-CELL_LINE O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O

(e) A53T α-synuclein clearance in undifferentiated or nonmitotic neuronally differentiated (with 100 ng ml-1 nerve growth factor for 5 d) stable PC12 cells as in Figure 1a, treated with or without 10 μM calpastatin for 24 h. Densitometry is relative to actin. Error bars show s.e.m.


( f ) COS - 7 , transfected with EGFP - HDQ74 along with empty vector ( pcDNA3 . 1 ) or constitutive active ( CA ) m - calpain ( 1 : 3 ) for 48 h , were assessed for aggregation as in Figure 1c . Error bars show 95 % confidence interval .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O

(f) COS-7 cells, transfected with EGFP-HDQ74 along with empty vector (pcDNA3.1) or constitutive active (CA) m-calpain (1:3 ratio) for 48 h, were assessed for aggregation and cell death as in Figure 1c. Error bars show 95% confidence interval.


( g ) SK - N - SH , transfected with EGFP - HDQ74 along with empty vector ( pcDNA3 . 1 ) or constitutive active m - calpain ( 1 : 3 ) for 4 h , were subsequently treated with DMSO ( control ) , 1 μM verapamil or 1 μM clonidine for 48 h . proportions of EGFP - positive with were assessed as in Figure 1c . Error bars show 95 % confidence interval . * * * P 0 . 001 ; * * P 0 . 01 ; * P 0 . 05 .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(g) SK-N-SH cells, transfected with EGFP-HDQ74 along with empty vector (pcDNA3.1) or constitutive active m-calpain (1:3 ratio) for 4 h, were subsequently treated with DMSO (control), 1 μM verapamil or 1 μM clonidine for 48 h. The proportions of EGFP-positive cells with aggregates were assessed as in Figure 1c. Error bars show 95% confidence interval. ***P 0.001; **P 0.01; *P 0.05.


( a ) proportions of EGFP - positive MEFs with , transfected with EGFP - HDQ74 pcDNA3 . 1 ( empty vector ) in Atg5 + / + , or transfected with EGFP - HDQ74 pcDNA3 . 1 , WT Atg5 or K130R Atg5 constructs in Atg5 - / - for 4 h ( 1 : 3 ratios ) , were treated for next 48 h with or without 10 μM calpastatin . data are from same experiment , but odds ratios for reference conditions in specific experiments have been set to 1 , to facilitate comparisons . Atg5 - / - had increased EGFP - HDQ74 compared to Atg5 + / + . Error bars show 95 % confidence interval .

O O O O O B-GENEPROD O O B-CELL_LINE O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O

(a) The proportions of EGFP-positive MEFs with aggregates, transfected with EGFP-HDQ74 and pcDNA3.1 (empty vector) in Atg5+/+ cells, or transfected with EGFP-HDQ74 and pcDNA3.1, WT Atg5 or K130R Atg5 constructs in Atg5-/- cells for 4 h (1:3 ratios), were treated for next 48 h with or without 10 μM calpastatin. All data are from the same experiment, but odds ratios for reference conditions in specific experiments have been set to 1, to facilitate comparisons. Atg5-/- cells had increased EGFP-HDQ74 aggregates compared to Atg5+/+ cells. Error bars show 95% confidence interval.


( b ) HeLa were transfected with siGLO ( control ) with or without calpain 1 siRNA or calpain 2 siRNA ( 1 : 3 ) for 96 h , followed by transfection with EGFP - LC3 construct for 4 h . were fixed after a further 2 h . proportions of transfected with > 5 EGFP - LC3 vesicles were assessed . Error bars show 95 % confidence interval .

O O O B-CELL_LINE O O O O O O O O O O B-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O

(b) HeLa cells were transfected with siGLO (control) with or without calpain 1 siRNA or calpain 2 siRNA (1:3 ratio) for 96 h, followed by transfection with EGFP-LC3 construct for 4 h. Cells were fixed after a further 2 h. The proportions of transfected cells with >5 EGFP-LC3 vesicles were assessed. Error bars show 95% confidence interval.


( c ) Endogenous LC3 - II in SK - N - SH treated with or without 10 μM or 30 μM calpastatin for 24 h .

O O O O B-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O B-GENEPROD O O O O

(c) Endogenous LC3-II levels in SK-N-SH cells treated with or without 10 μM or 30 μM calpastatin for 24 h.


( d ) HeLa stably expressing EGFP - LC3 were treated for 4 h with 400 nM bafilomycin A1 in presence or absence of 10 μM calpastatin , 50 μM ALLM or 50 μM calpeptin . Calpain inhibitors were treated for 24 h before bafilomycin A1 addition . EGFP - LC3 - II was detected with anti - EGFP antibody . Data from different calpain inhibitors are from different blots .

O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O

(d) HeLa cells stably expressing EGFP-LC3 were treated for 4 h with 400 nM bafilomycin A1 in presence or absence of 10 μM calpastatin, 50 μM ALLM or 50 μM calpeptin. Calpain inhibitors were treated for 24 h before bafilomycin A1 addition. EGFP-LC3-II was detected with anti-EGFP antibody. Data from different calpain inhibitors are from different blots.


( e ) COS - 7 , transfected with myc - LC3 EGFP - C1 with pcDNA3 . 1 ( empty vector ) or constitutively active ( CA ) m - calpain ( 1 : 1 : 3 ) for 4 h , were analyzed after 24 h post - transfection for LC3 - II by immunoblotting with anti - myc antibody . Error bars show s . e . m .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O

(e) COS-7 cells, transfected with myc-LC3 and EGFP-C1 with pcDNA3.1 (empty vector) or constitutively active (CA) m-calpain (1:1:3 ratio) for 4 h, were analyzed after 24 h post-transfection for LC3-II levels by immunoblotting with anti-myc antibody. Error bars show s.e.m.


( f ) COS - 7 , transfected with EGFP - LC3 pcDNA3 . 1 ( empty vector ) or CA m - calpain ( 1 : 3 ) for 4 h fixed 24 h post - transfection , were assessed for proportions of EGFP - positive with > 5 EGFP - LC3 vesicles . Error bars show 95 % confidence interval . * * * P 0 . 001 ; * * P 0 . 01 ; * P 0 . 05 ; NS , nonsignificant .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(f) COS-7 cells, transfected with EGFP-LC3 and pcDNA3.1 (empty vector) or CA m-calpain (1:3 ratio) for 4 h and fixed 24 h post-transfection, were assessed for proportions of EGFP-positive cells with >5 EGFP-LC3 vesicles. Error bars show 95% confidence interval. ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.


( a ) A53T α - synuclein clearance in stable PC12 as in Figure 1a , treated with or without 100 nM PACAP , with or without 10 μM calpastatin or 500 μM 2 ′ 5 ′ ddA for 24 h .

O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_LINE O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O

(a) A53T α-synuclein clearance in stable PC12 cells as in Figure 1a, treated with or without 100 nM PACAP, with or without 10 μM calpastatin or 500 μM 2′5′ddA for 24 h.


( b ) proportion of EGFP - positive with in SK - N - SH as in Figure 1c , treated for 48 h with 1 μM PACAP or 200 μM NF449 . Error bars show 95 % confidence interval .

O O O O O B-GENEPROD O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O

(b) The proportion of EGFP-positive cells with aggregates in SK-N-SH cells as in Figure 1c, treated for 48 h with 1 μM PACAP or 200 μM NF449. Error bars show 95% confidence interval.


( c ) Aggregation in COS - 7 transfected with either pcDNA3 : 1 ( empty vector ) or constitutively active ( CA ) m - calpain EGFP - HDQ74 ( 3 : 1 ) for 4 h then treated with or without 500 μM 2 ′ 5 ′ ddA for 48 h . Error bars show 95 % confidence interval .

O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O

(c) Aggregation in COS-7 cells transfected with either pcDNA3:1 (empty vector) or constitutively active (CA) m-calpain and EGFP-HDQ74 (3:1 ratio) for 4 h and then treated with or without 500 μM 2′5′ddA for 48 h. Error bars show 95% confidence interval.


( d ) HeLa transfected with control or Gsα siRNA for 72 h were analyzed for Gsα by immunoblotting with anti - Gsα antibody . Aggregation was assessed in HeLa transfected with control or Gsα siRNA EGFP - HDQ74 for 72 h . Error bars show 95 % confidence interval .

O O O B-CELL_LINE O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-EXP_ASSAY O O O B-GENEPROD O O O O O O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O

(d) HeLa cells transfected with control or Gsα siRNA for 72 h were analyzed for Gsα levels by immunoblotting with anti-Gsα antibody. Aggregation was assessed in HeLa cells transfected with control or Gsα siRNA and EGFP-HDQ74 for 72 h. Error bars show 95% confidence interval.


( e ) Endogenous LC3 - II in HeLa transfected with control or Gsα siRNA for 72 h .

O O O O B-GENEPROD O O O B-CELL_LINE O O O O B-GENEPROD O O O O O

(e) Endogenous LC3-II levels in HeLa cells transfected with control or Gsα siRNA for 72 h.


( f ) Endogenous LC3 - II in PC12 treated with or without 200 μM NF449 for 24 h .

O O O O B-GENEPROD O O O B-CELL_LINE O O O O O O B-SMALL_MOLECULE O O O O

(f) Endogenous LC3-II levels in PC12 cells treated with or without 200 μM NF449 for 24 h.


( g ) Endogenous LC3 - II in HeLa transfected with control or Gsα siRNA for 72 h treated with or without 400 nM bafilomycin A1 for last 4 h . Densitometric is relative to actin . Error bars show s . e . m .

O O O O B-GENEPROD O O O B-CELL_LINE O O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O O O O O O O O

(g) Endogenous LC3-II levels in HeLa cells transfected with control or Gsα siRNA for 72 h and treated with or without 400 nM bafilomycin A1 for the last 4 h. Densitometric analysis is relative to actin. Error bars show s.e.m.


( h ) proportions of EGFP - positive HeLa with , transfected with control or Gsα siRNA for 48 h , then retransfected together with pcDNA 3 . 1 ( empty vector ) or CA m - calpain EGFP - HDQ74 ( 3 : 1 ) for a further 48 h . Error bars show 95 % confidence interval . * * * P 0 . 001 ; * * P 0 . 01 ; * P 0 . 05 ; NS , nonsignificant .

O O O O O B-GENEPROD O O B-CELL_LINE O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(h) The proportions of EGFP-positive HeLa cells with aggregates, transfected with control or Gsα siRNA for 48 h, and then retransfected together with pcDNA 3.1 (empty vector) or CA m-calpain and EGFP-HDQ74 (3:1 ratio) for a further 48 h. Error bars show 95% confidence interval. ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.


( a ) proportions of EGFP - positive COS - 7 with or , after transfection with EGFP - HDQ74 rheb or pcDNA3 . 1 ( empty vector ) ( 1 : 3 ) for 4 h treatment with or without 10 μM calpastatin or 50 μM calpeptin for 48 h post - transfection . control condition for assessing effect of calpain inhibitors in rheb - transfected was taken as 1 . Error bars show 95 % confidence interval .

O O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O

(a) The proportions of EGFP-positive COS-7 cells with aggregates or cell death, after transfection with EGFP-HDQ74 and rheb or pcDNA3.1 (empty vector) (1:3 ratio) for 4 h and treatment with or without 10 μM calpastatin or 50 μM calpeptin for 48 h post-transfection. The control condition for assessing the effect of calpain inhibitors in rheb-transfected cells was taken as 1. Error bars show 95% confidence interval.


( b ) proportions of EGFP - positive COS - 7 with > 5 EGFP - LC3 vesicles , transfected with EGFP - LC3 pcDNA3 . 1 ( empty vector ) or rheb ( 1 : 3 ) for 4 h treated with or without 10 μM calpastatin or 50 μM calpeptin for 24 h post - transfection . Error bars show 95 % confidence interval .

O O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O

(b) The proportions of EGFP-positive COS-7 cells with >5 EGFP-LC3 vesicles, transfected with EGFP-LC3 and pcDNA3.1 (empty vector) or rheb (1:3 ratio) for 4 h and treated with or without 10 μM calpastatin or 50 μM calpeptin for 24 h post-transfection. Error bars show 95% confidence interval.


( c ) proportions of EGFP - positive COS - 7 with , transfected with EGFP - HDQ74 constitutively active ( CA ) m - calpain or pcDNA3 . 1 ( empty vector ) ( 1 : 3 ) for 4 h treated with or without 0 . 2 μM rapamycin for 48 h post - transfection . Error bars show 95 % confidence interval .

O O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O

(c) The proportions of EGFP-positive COS-7 cells with aggregates, transfected with EGFP-HDQ74 and constitutively active (CA) m-calpain or pcDNA3.1 (empty vector) (1:3 ratio) for 4 h and treated with or without 0.2 μM rapamycin for 48 h post-transfection. Error bars show 95% confidence interval.


( d ) proportions of EGFP - positive COS - 7 with > 5 EGFP - LC3 vesicles , transfected with EGFP - LC3 CA m - calpain or pcDNA3 . 1 ( empty vector ) ( 1 : 3 ) for 4 h treated with or without 0 . 2 μM rapamycin for 24 h post - transfection . Error bars show 95 % confidence interval .

O O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O

(d) The proportions of EGFP-positive COS-7 cells with >5 EGFP-LC3 vesicles, transfected with EGFP-LC3 and CA m-calpain or pcDNA3.1 (empty vector) (1:3 ratio) for 4 h and treated with or without 0.2 μM rapamycin for 24 h post-transfection. Error bars show 95% confidence interval.


( e , f ) proportions of EGFP - positive COS - 7 with ( e ) ( f ) as in Figure 1c , treated with or without 0 . 2 μM rapamycin , 10 μM calpastatin or both for 48 h . Error bars show 95 % confidence interval .

O O O O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O O O O O O O O O O

(e,f) The proportions of EGFP-positive COS-7 cells with aggregates (e) and cell death (f) as in Figure 1c, treated with or without 0.2 μM rapamycin, 10 μM calpastatin or both for 48 h. Error bars show 95% confidence interval.


( g ) Clearance of soluble EGFP - HDQ74 in stable PC12 as in Figure 1b , treated with or without 0 . 2 μM rapamycin , 10 μM calpastatin or both for 48 h switch - off period . Error bars show s . e . m .

O O O O O O B-GENEPROD O O O O B-CELL_LINE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O

(g) Clearance of soluble EGFP-HDQ74 in stable PC12 cells as in Figure 1b, treated with or without 0.2 μM rapamycin, 10 μM calpastatin or both for the 48 h switch-off period. Error bars show s.e.m.


( h ) Clearance of A53T α - synuclein in stable PC12 as in Figure 1a , treated with or without 0 . 2 μM rapamycin , 10 μM calpastatin or both for 8 h switch - off period . Error bars show s . e . m . * * * P 0 . 001 ; * * P 0 . 01 ; * P 0 . 05 ; NS , nonsignificant .

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_LINE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(h) Clearance of A53T α-synuclein in stable PC12 cells as in Figure 1a, treated with or without 0.2 μM rapamycin, 10 μM calpastatin or both for the 8 h switch-off period. Error bars show s.e.m. ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.


( a ) Western blot showing EGFP - HDQ71 - induced degeneration of rod photoreceptors at 9 d . p . f . as indicated by decrease in abundance of of monomeric ( ∼ 36 kDa ) dimeric ( ∼ 72 kDa ) rhodopsin . Rhodopsin expression is further reduced by 3 μM ( ± ) - Bay K8644 , recovered upon treatment with 3 μM verapamil , 100 μM 2 ′ 5 ′ ddA , 3 μM clonidine 1 μM calpastatin . No general toxicity to zebrafish at these concentrations was observed . ( b ) Expression of EGFP - HDQ71 significantly reduces of rhodopsin expression when compared to wild - type ( WT ) nontransgenic controls . This effect is further abrogated by 3 μM ( ± ) - Bay K8644 , whereas 3 μM verapamil , 100 μM 2 ′ 5 ′ ddA , 3 μM clonidine 1 μM calpastatin protected against rod photoreceptor degeneration . Error bars show s . e . m .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O

(a) Western blot showing EGFP-HDQ71-induced degeneration of rod photoreceptors at 9 d.p.f. as indicated by the decrease in the abundance of of monomeric (∼36 kDa) and dimeric (∼72 kDa) rhodopsin. Rhodopsin expression is further reduced by 3 μM (±)-Bay K8644, and recovered upon treatment with 3 μM verapamil, 100 μM 2′5′ddA, 3 μM clonidine and 1 μM calpastatin. No general toxicity to the zebrafish at these concentrations was observed. (b) Expression of EGFP-HDQ71 significantly reduces the levels of rhodopsin expression when compared to wild-type (WT) nontransgenic controls. This effect is further abrogated by 3 μM (±)-Bay K8644, whereas 3 μM verapamil, 100 μM 2′5′ddA, 3 μM clonidine and 1 μM calpastatin all protected against rod photoreceptor degeneration. Error bars show s.e.m.


( c ) Magnified bright field EGFP of zebrafish eye sections , taken at ventral marginal zone for each of indicated treatments ( with concentrations as in b ) , are shown . Scale bar , 10 μm . * * * P 0 . 001 ; * * P 0 . 01 ; * P 0 . 05 . ( d ) total number of across 8 × 12 μm sections was counted compared to EGFP - HDQ71 DMSO - treated control . 3 μM ( ± ) - Bay K8644 was found to significantly increase aggregate burden , whereas 3 μM verapamil , 1 μM calpastatin , 3 μM clonidine 100 μM 2 ′ 5 ′ ddA decreased aggregate load . As in our models , these were insoluble in 4 % triton / SDS . Error bars show s . e . m . * * * P 0 . 001 ; * * P 0 . 01 ; * P 0 . 05 .

O O O O O O B-GENEPROD O B-ORGANISM B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) Magnified bright field and EGFP images of zebrafish eye sections, taken at the ventral marginal zone for each of the indicated treatments (with concentrations as in b), are shown. Scale bar, 10 μm. ***P 0.001; **P 0.01; *P 0.05. (d) The total number of aggregates across 8 × 12 μm sections was counted and compared to the EGFP-HDQ71 DMSO-treated control. 3 μM (±)-Bay K8644 was found to significantly increase the aggregate burden, whereas 3 μM verapamil, 1 μM calpastatin, 3 μM clonidine and 100 μM 2′5′ddA decreased the aggregate load. As in our cell models, these aggregates were insoluble in 4% triton/SDS. Error bars show s.e.m. ***P 0.001; **P 0.01; *P 0.05.


B : CL formation was measured in presence of 60 nmol MLCL ( 18 : 2 ) 3 , 40 nmol PC ( 18 : 2 ) 2 , 4 µg TAZ , 0 . 2 mg MBP or 0 . 2 mg Alb . C : CL formation was measured as in B with different amounts of MBP or Alb .

O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O

B: CL formation was measured in the presence of 60 nmol MLCL(18:2)3, 40 nmol PC(18:2)2, 4 µg TAZ, and 0.2 mg MBP or 0.2 mg Alb. C: CL formation was measured as in B with different amounts of MBP or Alb.


G : CL formation was measured in presence of 40 nmol PC ( 18 : 2 ) 2 , 10 nmol MLCL ( 18 : 2 ) 3 , 8 µg TAZ , 1250 nmol Triton X - 100 ( TX ) , 5 . 6 nmol bacteriorhodopsin ( BR ) . H : PC formation was measured in presence of 180 nmol CL ( 18 : 2 ) 4 , 20 nmol LPC16 : 0 , 8 µg TAZ , 20 mM Ca2 + , 5 . 6 nmol BR .

O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-GENEPROD O

G: CL formation was measured in the presence of 40 nmol PC(18:2)2, 10 nmol MLCL(18:2)3, 8 µg TAZ, 1250 nmol Triton X-100 (TX), and 5.6 nmol bacteriorhodopsin (BR). H: PC formation was measured in the presence of 180 nmol CL(18:2)4, 20 nmol LPC16:0, 8 µg TAZ, 20 mM Ca2+, and 5.6 nmol BR.


J : Transacylations were measured as in I in presence of 0 . 9 g BR / g lipid . of total linoleoyl transfer over total oleoyl transfer was calculated .

O O B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O

J: Transacylations were measured as in I in the presence of 0.9 g BR/ g lipid. The ratio of total linoleoyl transfer over total oleoyl transfer was calculated.


A : Inner ( IM ) outer ( OM ) mitochondrial membranes were traced in randomly collected electron in order to calculate IM / OM surface . Multiple were analyzed per biological replica .

O O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-EXP_ASSAY O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O

A: Inner (IM) and outer (OM) mitochondrial membranes were traced in randomly collected electron micrographs in order to calculate the IM/OM surface area ratio. Multiple micrographs were analyzed per biological replica.


A : Mitochondrial membranes were traced in electron to determine IM / OM surface . Tracings of IM are shown in yellow boxes .

O O B-SUBCELLULAR I-SUBCELLULAR O O O B-EXP_ASSAY O O B-SUBCELLULAR O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O

A: Mitochondrial membranes were traced in electron micrographs to determine the IM/OM surface area ratio. Tracings of the IM are shown in yellow boxes.


C : abundances were measured in adult hearts by quantitative Western blotting with - labeled secondary antibodies . abundances of IM ( NDUFB6 , UQCR2 , ATP5A1 ) are expressed relative to abundance of OM TOM70 .

O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-SUBCELLULAR B-GENEPROD O

C: Protein abundances were measured in adult hearts by quantitative Western blotting with fluorescence-labeled secondary antibodies. The abundances of IM proteins (NDUFB6, UQCR2, ATP5A1) are expressed relative to the abundance of the OM protein TOM70.


Tissues were analyzed by quantitative electron microscopy . Multiple were analyzed per biological

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O

Tissues were analyzed by quantitative electron microscopy. Multiple micrographs were analyzed per biological replica


F , G : Tissues were analyzed by label - free quantitative proteomics . C I - V , I - V ; CAC , citric acid cycle enzymes ; MICOS , contact site ; mt TA , mitochondrial transaminases ; NS , not significant ; PDH pyruvate dehydrogenase ; VDAC , voltage - dependent anion channel

O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD

F, G: Tissues were analyzed by label-free quantitative proteomics. C I-V, complex I-V; CAC, citric acid cycle enzymes; MICOS, contact site complex; mt TA, mitochondrial transaminases; NS, not significant; PDH pyruvate dehydrogenase; VDAC, voltage-dependent anion channel


A . Immunofluorescence of SATB2 ( green ) GSC marker SOX2 or OLIG2 ( red ) on frozen tissue sections of human GBM surgical specimens . SATB2 is preferentially expressed by GSCs in human GBMs . Scale bar , 25 μm . B . of fraction of SATB2 + in SOX2 + or OLIG2 + in human GBMs . More than 90 % SOX2 + or OLIG2 + showed SATB2 . n = 3 GBMs .

O O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-ORGANISM B-DISEASE O O O B-GENEPROD O O O O O O B-ORGANISM B-DISEASE O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-ORGANISM B-DISEASE O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O B-DISEASE O

A. Immunofluorescence of SATB2 (green) and the GSC marker SOX2 or OLIG2 (red) on frozen tissue sections of human GBM surgical specimens. SATB2 is preferentially expressed by GSCs in human GBMs. Scale bar, 25 μm. B. Quantification of the fraction of SATB2+ cells in SOX2+ or OLIG2+ cells in human GBMs. More than 90% SOX2+ or OLIG2+ cells showed SATB2 staining. n=3 GBMs.


C . qPCR of SATB2 mRNA expression in GSCs matched non - ( NSTCs ) ( n = 5 ) .

O O B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O

C. qPCR analysis of SATB2 mRNA expression in GSCs and matched non-stem tumor cells (NSTCs) (n=5).


D . Immunoblot of SATB2 , SOX2 OLIG2 expression in lysates of GSCs matched NSTCs .

O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O

D. Immunoblot analysis of SATB2, SOX2 and OLIG2 expression in cell lysates of GSCs and matched NSTCs.


E . Immunofluorescenc of SATB2 ( green ) SOX2 ( red ) in T3359 GSCs matched NSTCs . Scale bar , 50 μm .

O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O

E. Immunofluorescenc of SATB2 (green) and SOX2 (red) in T3359 GSCs and matched NSTCs. Scale bar, 50 μm.


F . qPCR of SATB2 mRNA expression in GSCs neural progenitor ( NPCs ) ( n = 3 ) .

O O B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O

F. qPCR analysis of SATB2 mRNA expression in GSCs and neural progenitor cells (NPCs) (n=3).


G . Immunoblot of SATB2 SOX2 expression in lysates of GSCs NPCs .

O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O

G. Immunoblot analysis of SATB2 and SOX2 expression in cell lysates of GSCs and NPCs.


H . Immunoblot of SATB2 , GSC marker SOX2 differentiation marker GFAP expression during serum - induced GSC differentiation .

O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD B-EXP_ASSAY O B-TISSUE O O O O O

H. Immunoblot analysis of SATB2, GSC marker SOX2 and differentiation marker GFAP expression during serum-induced GSC differentiation.


A . Immunoblot of SATB2 expression in GSCs transduced with lentiviral - mediated non - targeting shRNA ( shNT ) or SATB2 shRNA ( shSATB2 ) .

O O B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O O O B-ORGANISM O O O O O O O O O O B-GENEPROD O O O O O

A. Immunoblot analysis of SATB2 expression in GSCs transduced with lentiviral-mediated non-targeting shRNA (shNT) or SATB2 shRNA (shSATB2).


B . viability of GSCs transduced with shNT or shSATB2 ( n = 5 ) .

O O B-EXP_ASSAY O O O O O O O O O O O O O

B. Cell viability of GSCs transduced with shNT or shSATB2 (n=5).


C . EdU incorporation of GSCs transduced with shNT or shSATB2 . Scale bar : 50 μm . D . of ( C ) showing of EdU + ( n = 5 ) .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O

C. EdU incorporation assay of GSCs transduced with shNT or shSATB2. Scale bar: 50 μm. D. Quantification of (C) showing the percentage of EdU+ cells (n=5).


E . Tumorsphere of GSCs transduced with shNTor shSATB2 . Scale bar : 100 μm . of diameter of tumorspheres formed by GSCs expressing shNT or shSATB2 ( F : n = 9 ; G : n = 5 ) .

O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O

E. Tumorsphere images of GSCs transduced with shNTor shSATB2. Scale bar: 100 μm. Quantification of the diameter of tumorspheres formed by GSCs expressing shNT or shSATB2 (F: n=9; G: n=5).


G . of number of tumorspheres formed by GSCs expressing shNT or shSATB2 ( F : n = 9 ; G : n = 5 ) .

O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O

G. Quantification of the number of tumorspheres formed by GSCs expressing shNT or shSATB2 (F: n=9; G: n=5).


H . In vitro limiting dilution of tumorsphere formations of GSCs expressing shNT or shSATB2 . Silencing SATB2 attenuated self - renewal capacity of GSCs .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O

H. In vitro limiting dilution analysis of the tumorsphere formations of GSCs expressing shNT or shSATB2. Silencing SATB2 attenuated the self-renewal capacity of GSCs.


A . Bioluminescent of GBM xenografts derived from luciferase - labeled T3359 GSCs expressing NT or SATB2 shRNA . Representative on day 21 posttransplantation are shown ( n = 5 mice per group ) . Silencing SATB2 significantly delayed GBM growth .

O O B-EXP_ASSAY O B-DISEASE B-EXP_ASSAY O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-ORGANISM O O O O O B-GENEPROD O O B-DISEASE B-EXP_ASSAY O

A. Bioluminescent images of the GBM xenografts derived from the luciferase-labeled T3359 GSCs expressing NT or SATB2 shRNA. Representative images on day 21 posttransplantation are shown (n=5 mice per group). Silencing SATB2 significantly delayed GBM growth .


A . Bioluminescent of GBM xenografts derived from luciferase - labeled T3359 GSCs expressing NT or SATB2 shRNA . Representative on day 21 posttransplantation are shown ( n = 5 mice per group ) . Silencing SATB2 significantly delayed GBM

O O B-EXP_ASSAY O B-DISEASE B-EXP_ASSAY O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-ORGANISM O O O O O B-GENEPROD O O B-DISEASE

A. Bioluminescent images of the GBM xenografts derived from the luciferase-labeled T3359 GSCs expressing NT or SATB2 shRNA. Representative images on day 21 posttransplantation are shown (n=5 mice per group). Silencing SATB2 significantly delayed GBM growth


C . Kaplan - Meier survival curves of mice intracranially implanted with T3359 GSCs expressing shNT or shSATB2 ( shNT : n = 7 mice ; shSATB2 - 1 or shSATB2 - 2 : n = 5 mice ) . Median survival : shNT , 28 days ; shSATB2 - 1 , 48 days ; shSATB2 - 2 , 45 Days . bearing GSC - derived xenografts expressing SATB2 shRNA survived longer than control .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O B-ORGANISM O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O

C. Kaplan-Meier survival curves of mice intracranially implanted with T3359 GSCs expressing shNT or shSATB2 (shNT: n=7 mice; shSATB2-1 or shSATB2-2: n=5 mice). Median survival: shNT, 28 days; shSATB2-1, 48 days; shSATB2-2, 45 Days. Animals bearing GSC-derived xenografts expressing SATB2 shRNA survived longer than the control animals.


D . Immunofluorescence of SATB2 ( Green ) in xenografts derived from T3359 GSCs expressing shNT or shSATB2 ( n = 5 tumors per group ) . Scale bar : 40 μm . E . of SATB2 in xenografts derived from T3359 GSCs expressing shNT or shSATB2 ( n = 5 tumors per group ) .

O O B-EXP_ASSAY O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O O O O O O

D. Immunofluorescence of SATB2 (Green) in xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n=5 tumors per group). Scale bar: 40 μm. E. Quantification of SATB2 intensity in xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n=5 tumors per group).


D . Immunofluorescence of SATB2 ( Green ) in xenografts derived from T3359 GSCs expressing shNT or shSATB2 ( n = 5 tumors per group ) . Scale bar : 40 μm .

O O B-EXP_ASSAY O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O

D. Immunofluorescence of SATB2 (Green) in xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n=5 tumors per group). Scale bar: 40 μm.


F . Immunofluorescence of Ki67 ( Green ) in xenografts derived from T3359 GSCs expressing shNT or shSATB2 ( n = 5 tumors per group ) . Scale bar : 40 μm . G . of Ki67 positive in xenografts derived from T3359 GSCs expressing shNT or shSATB2 ( n = 5 tumors per group ) .

O O B-EXP_ASSAY O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O

F. Immunofluorescence of Ki67 (Green) in tumor xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n=5 tumors per group). Scale bar: 40 μm. G. Quantification of Ki67 positive cells in xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n=5 tumors per group).


F . Immunofluorescence of Ki67 ( Green ) in xenografts derived from T3359 GSCs expressing shNT or shSATB2 ( n = 5 tumors per group ) . Scale bar : 40 μm .

F. Immunofluorescence of Ki67 (Green) in tumor xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n=5 tumors per group). Scale bar: 40 μm.


H . Immunofluorescence of SOX2 ( Red ) in xenografts derived from T3359 GSCs expressing shNT or shSATB2 ( n = 4 tumors per group ) . Scale bar : 40 μm . I . of SOX2 positive in xenografts derived from T3359 GSCs expressing shNT or shSATB2 ( n = 4 tumors per group ) .

H. Immunofluorescence of SOX2 (Red) in xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n=4 tumors per group). Scale bar: 40 μm. I. Quantification of SOX2 positive cells in xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n=4 tumors per group).


H . Immunofluorescence of SOX2 ( Red ) in xenografts derived from T3359 GSCs expressing shNT or shSATB2 ( n = 4 tumors per group ) . Scale bar : 40 μm .

H. Immunofluorescence of SOX2 (Red) in xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n=4 tumors per group). Scale bar: 40 μm.


A . of differentially expressed genes between SATB2 silencing H2S GSCs ( shSATB2 ) control H2S GSCs ( shNT ) . Differentially expressed genes had a 1 . 8 - fold or greater expression difference . Among differentially expressed genes , 160 are upregulated 185 are downregulated .

O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O

A. Heatmap analysis of differentially expressed genes between SATB2 silencing H2S GSCs (shSATB2) and control H2S GSCs (shNT). Differentially expressed genes had a 1.8-fold or greater expression difference. Among differentially expressed genes, 160 are upregulated and 185 are downregulated.


C . qPCR of FOXM1 SATB2 mRNA expression in GSCs transduced with shNT or shSATB2 ( n = 3 ) .

O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O

C. qPCR analysis of FOXM1 and SATB2 mRNA expression in GSCs transduced with shNT or shSATB2 (n=3).


D . Immunoblot of FOXM1 SATB2 expression in GSCs transduced with shNT or shSATB2 .

O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O

D. Immunoblot analysis of FOXM1 and SATB2 expression in GSCs transduced with shNT or shSATB2.


E . Immunoblot of FOXM1 SATB2 expression in xenografts - derived from T3359 GSCs expressing shNT or shSATB2 .

O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O

E. Immunoblot analysis of FOXM1 and SATB2 expression in tumor xenografts-derived from T3359 GSCs expressing shNT or shSATB2.


F . of FOXM1 downstream targets between SATB2 silencing H2S GSCs ( shSATB2 ) control H2S GSCs ( shNT ) from microarray . Differentially expressed genes had a 1 . 5 - fold or greater expression difference .

O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY O O

F. Heatmap analysis of FOXM1 downstream targets between SATB2 silencing H2S GSCs (shSATB2) and control H2S GSCs (shNT) from microarray analysis. Differentially expressed genes had a 1.5-fold or greater expression difference.


G . qPCR of FOXM1 downstream targets in H2S GSCs transduced with shNT or shSATB2 ( n = 3 ) .

O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O

G. qPCR analysis of FOXM1 downstream targets in H2S GSCs transduced with shNT or shSATB2 (n=3).


A . Immunoblot of FOXM1 SATB2 expression in T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 .

O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O

A. Immunoblot analysis of FOXM1 and SATB2 expression in T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2.


B . viability of T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 ( n = 5 ) . Ectopic expression of FOXM1 restored proliferation impaired by SATB2 silencing .

O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O O B-GENEPROD O O

B. Cell viability assay of T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (n=5). Ectopic expression of FOXM1 restored the cell proliferation impaired by SATB2 silencing.


C . Tumorsphere number of T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 ( n = 5 ) . Ectopic expression of FOXM1 restored tumorsphere formation of GSCs impaired by SATB2 silencing .

O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O

C. Tumorsphere number of T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (n=5). Ectopic expression of FOXM1 restored the tumorsphere formation of GSCs impaired by SATB2 silencing.


In vivo bioluminescent of xenografts derived from luciferase - labeled T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 ( shNT : n = 5 mice ; shNT + FOXM1 : n = 4 mice ; shSATB2 : n = 5 mice ; shSATB2 + FOXM1 : n = 5 mice ) . Representative on day 21 posttransplantation are shown . Ectopic expression of FOXM1 in GSCs expressing shSATB2 markedly restored GBM growth .

O O B-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-ORGANISM O O O B-GENEPROD O O O O B-ORGANISM O O O O O O B-ORGANISM O O O B-GENEPROD O O O O B-ORGANISM O O O O O O O O O O O O O B-GENEPROD O O O O O O B-DISEASE B-EXP_ASSAY O

In vivo bioluminescent images of the tumor xenografts derived from luciferase-labeled T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n=5 mice; shNT+FOXM1: n=4 mice; shSATB2: n=5 mice; shSATB2+ FOXM1: n=5 mice). Representative images on day 21 posttransplantation are shown. Ectopic expression of FOXM1 in GSCs expressing shSATB2 markedly restored GBM tumor growth.


E . In vivo bioluminescent or of xenografts derived from luciferase - labeled T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 ( shNT : n = 5 mice ; shNT + FOXM1 : n = 4 mice ; shSATB2 : n = 5 mice ; shSATB2 + FOXM1 : n = 5 mice ) . Representative on day 21 posttransplantation are shown . Ectopic expression of FOXM1 in GSCs expressing shSATB2 markedly restored GBM growth .

O O O O B-EXP_ASSAY O O B-EXP_ASSAY O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-ORGANISM O O O B-GENEPROD O O O O B-ORGANISM O O O O O O B-ORGANISM O O O B-GENEPROD O O O O B-ORGANISM O O O O O O O O O O O O O B-GENEPROD O O O O O O B-DISEASE B-EXP_ASSAY O

E. In vivo bioluminescent images or quantification of the tumor xenografts derived from luciferase-labeled T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n=5 mice; shNT+FOXM1: n=4 mice; shSATB2: n=5 mice; shSATB2+ FOXM1: n=5 mice). Representative images on day 21 posttransplantation are shown. Ectopic expression of FOXM1 in GSCs expressing shSATB2 markedly restored GBM tumor growth.


F . Kaplan - Meier survival curves of mice intracranially implanted with T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 ( shNT : n = 8 mice ; shNT + FOXM1 : n = 7 mice ; shSATB2 : n = 6 mice ; shSATB2 + FOXM1 : n = 8 mice ) . Median survival : shNT , 30 days ; shNT + FOXM1 , 28 days ; shSATB2 , 50 . 5 Days ; shSATB2 + FOXM1 , 34 days . Ectopic expression of FOXM1 in GSCs expressing shSATB2 markedly attenuated increased survival of mice bearing GSC - derived GBMs .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-ORGANISM O O O B-GENEPROD O O O O B-ORGANISM O O O O O O B-ORGANISM O O O B-GENEPROD O O O O B-ORGANISM O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O B-ORGANISM O O O O B-DISEASE O

F. Kaplan-Meier survival curves of mice intracranially implanted with T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n=8 mice; shNT+FOXM1: n=7 mice; shSATB2: n=6 mice; shSATB2+ FOXM1: n=8 mice). Median survival: shNT, 30 days; shNT+FOXM1, 28 days; shSATB2, 50.5 Days; shSATB2+FOXM1, 34 days. Ectopic expression of FOXM1 in GSCs expressing shSATB2 markedly attenuated the increased survival of mice bearing the GSC-derived GBMs.


G . Immunofluorescence of Ki67 ( Green ) in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 ( shNT : n = 6 tumors ; shNT + FOXM1 : n = 5 tumors ; shSATB2 : n = 6 tumors ; shSATB2 + FOXM1 : n = 6 tumors ) . Scale bar : 40 μm . H . of Ki67 positive in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 ( shNT : n = 6 tumors ; shNT + FOXM1 : n = 5 tumors ; shSATB2 : n = 6 tumors ; shSATB2 + FOXM1 : n = 6 tumors ) .

O O B-EXP_ASSAY O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O

G. Immunofluorescence of Ki67 (Green) in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n=6 tumors; shNT+FOXM1: n=5 tumors; shSATB2: n=6 tumors; shSATB2+ FOXM1: n=6 tumors). Scale bar: 40 μm. H. Quantification of Ki67 positive cells in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n=6 tumors; shNT+FOXM1: n=5 tumors; shSATB2: n=6 tumors; shSATB2+ FOXM1: n=6 tumors).


G . Immunofluorescence of Ki67 ( Green ) in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 ( shNT : n = 6 tumors ; shNT + FOXM1 : n = 5 tumors ; shSATB2 : n = 6 tumors ; shSATB2 + FOXM1 : n = 6 tumors ) . Scale bar : 40 μm .

O O B-EXP_ASSAY O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O

G. Immunofluorescence of Ki67 (Green) in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n=6 tumors; shNT+FOXM1: n=5 tumors; shSATB2: n=6 tumors; shSATB2+ FOXM1: n=6 tumors). Scale bar: 40 μm.


I . Immunofluorescence of SOX2 ( Red ) in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 ( shNT : n = 6 tumors ; shNT + FOXM1 : n = 5 tumors ; shSATB2 : n = 6 tumors ; shSATB2 + FOXM1 : n = 6 tumors ) . Scale bar : 40 μm . J . of SOX2 positive in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 ( shNT : n = 6 tumors ; shNT + FOXM1 : n = 5 tumors ; shSATB2 : n = 6 tumors ; shSATB2 + FOXM1 : n = 6 tumors ) .

I. Immunofluorescence of SOX2 (Red) in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n=6 tumors; shNT+FOXM1: n=5 tumors; shSATB2: n=6 tumors; shSATB2+ FOXM1: n=6 tumors). Scale bar: 40 μm. J. Quantification of SOX2 positive cells in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n=6 tumors; shNT+FOXM1: n=5 tumors; shSATB2: n=6 tumors; shSATB2+ FOXM1: n=6 tumors).


I . Immunofluorescence of SOX2 ( Red ) in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 ( shNT : n = 6 tumors ; shNT + FOXM1 : n = 5 tumors ; shSATB2 : n = 6 tumors ; shSATB2 + FOXM1 : n = 6 tumors ) . Scale bar : 40 μm .

I. Immunofluorescence of SOX2 (Red) in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n=6 tumors; shNT+FOXM1: n=5 tumors; shSATB2: n=6 tumors; shSATB2+ FOXM1: n=6 tumors). Scale bar: 40 μm.


B . ChIP assays with SATB2 antibody or IgG using GSCs , NSTCs NPCs . PCR primers amplified a fragment flanking MAR of FOXM1 gene locus . Note that abundant SATB2 binds to MAR of FOXM1 gene locus in GSCs .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O B-GENEPROD B-EXP_ASSAY O O O B-GENEPROD O O O O O

B. ChIP assays with the SATB2 antibody or IgG using GSCs, NSTCs and NPCs. PCR primers amplified a fragment flanking the MAR of FOXM1 gene locus. Note that abundant SATB2 binds to the MAR of FOXM1 gene locus in GSCs.


C . qPCR of ChIP assays with SATB2 antibody or IgG using GSCs transduced with shNT or shSATB2 ( n = 3 ) . PCR primers amplified a fragment flanking MAR of FOXM1 gene locus . Silencing SATB2 decreased its binding amount to MAR of FOXM1 gene locus .

O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O B-GENEPROD O O B-EXP_ASSAY O O O O B-GENEPROD O O O

C. qPCR analysis of ChIP assays with the SATB2 antibody or IgG using GSCs transduced with shNT or shSATB2 (n=3). PCR primers amplified a fragment flanking the MAR of FOXM1 gene locus. Silencing SATB2 decreased its binding amount to the MAR of FOXM1 gene locus.