Aβ profiles ( % contribution of individual Aβ37 , Aβ38 , Aβ40 , and Aβ42 peptides to the total Aβ levels ( Aβ37 + 38 + 40 + 42 ) ) in the conditioned medium collected from vehicle ( DMSO ) or GSM - treated ( 1 µM final concentration ) MEF cells co - expressing respective WT or mutant NCT GSEC with APPC99 were determined by ELISA . Data generated using 0 . 3 µM GSM - treated MEF cells is shown in Figure EV4 . Data are presented as mean ± SD , N ≥ 3 independent experiments .

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Aβ profiles (% contribution of individual Aβ37, Aβ38, Aβ40, and Aβ42 peptides to the total Aβ levels (Aβ37+38+40+42)) in the conditioned medium collected from vehicle (DMSO) or GSM-treated (1 µM final concentration) MEF cells co-expressing respective WT or mutant NCT GSEC with APPC99 were determined by ELISA. Data generated using 0.3 µM GSM-treated MEF cells is shown in Figure EV4. Data are presented as mean ± SD, N ≥ 3 independent experiments.


Aβ 37 / 40 and Aβ 38 / 42 ratios were calculated to evaluate the efficiency of the GSEC - mediated cleavage of APPC99 ( Figure EV4 ) and statistical analysis ( P values ) ( two - way ANOVA with Sidak ' s post hoc test ) for the effect of GSMs on WT and mutant complexes is presented ( response to GSM normalized to the corresponding vehicle condition ) .

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Aβ 37/40 and Aβ 38/42 ratios were calculated to evaluate the efficiency of the GSEC-mediated cleavage of APPC99 (Figure EV4) and statistical analysis (P values) (two-way ANOVA with Sidak's post hoc test) for the effect of GSMs on WT and mutant complexes is presented (response to GSM normalized to the corresponding vehicle condition).


A Immunoblot analysis of HeLa cells using the indicated antibodies that were synchronized in G1 / S phase using a double thymidine block or in mitosis using sequential thymidine and nocodazole treatment and collected at the given time points .

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A Immunoblot analysis of HeLa cells using the indicated antibodies that were synchronized in G1/S phase using a double thymidine block or in mitosis using sequential thymidine and nocodazole treatment and collected at the given time points.


B Immunoblot analysis of HeLa cells that were transfected with siRNA oligonucleotides as indicated and left untreated or synchronized in mitosis using sequential thymidine and nocodazole treatment .

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B Immunoblot analysis of HeLa cells that were transfected with siRNA oligonucleotides as indicated and left untreated or synchronized in mitosis using sequential thymidine and nocodazole treatment.


C Co - immunoprecipitation of XIAP and endogenous USP9X in HEK 293T cells that were transfected with FLAG - tagged XIAP or empty vector ( EV ) and synchronized in mitosis using nocodazole or left untreated .

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C Co-immunoprecipitation of XIAP and endogenous USP9X in HEK 293T cells that were transfected with FLAG-tagged XIAP or empty vector (EV) and synchronized in mitosis using nocodazole or left untreated.


D Co - immunoprecipitation of XIAP and endogenous USP9X in HEK 293T cells that were treated as in ( C ) , but with additional transfection of the FLAG - tagged XIAP single amino acid mutants G188R and G188E . Only mitotic samples are shown . The asterisk denotes ubiquitylated forms of XIAP .

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D Co-immunoprecipitation of XIAP and endogenous USP9X in HEK 293T cells that were treated as in (C), but with additional transfection of the FLAG-tagged XIAP single amino acid mutants G188R and G188E. Only mitotic samples are shown. The asterisk denotes ubiquitylated forms of XIAP.


E In vivo ubiquitylation of XIAP in HeLa cells that were infected with the indicated expression constructs carrying FLAG - tagged XIAP and transfected with siRNA oligonucleotides as specified . Cells were synchronized in mitosis using sequential thymidine / nocodazole treatment , as indicated . Subsequent to treatment with MG132 , whole cell extracts ( WCE ) were prepared and ubiquitylated XIAP was isolated by anti - FLAGimmunoprecipitation ( IP ) under denaturing conditions .

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E In vivo ubiquitylation of XIAP in HeLa cells that were infected with the indicated expression constructs carrying FLAG-tagged XIAP and transfected with siRNA oligonucleotides as specified. Cells were synchronized in mitosis using sequential thymidine/nocodazole treatment, as indicated. Subsequent to treatment with MG132, whole cell extracts (WCE) were prepared and ubiquitylated XIAP was isolated by anti-FLAGimmunoprecipitation (IP) under denaturing conditions.


F Immunoblot analysis of NIH 3T3 cells that were transfected with expression constructs for USP9XWT or the catalytically inactive mutant USP9XC1556S . The band in the EV control lane of the anti - V5 panel marks an unspecific band produced by the antibody .

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F Immunoblot analysis of NIH 3T3 cells that were transfected with expression constructs for USP9XWT or the catalytically inactive mutant USP9XC1556S. The band in the EV control lane of the anti-V5 panel marks an unspecific band produced by the antibody.


G Immunoblot analysis of HeLa cells using antibodies to the indicated endogenous proteins that were synchronized in mitosis using thymidine / nocodazole and treated with DMSO or the USP9X inhibitor WP1130 as indicated .

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G Immunoblot analysis of HeLa cells using antibodies to the indicated endogenous proteins that were synchronized in mitosis using thymidine/nocodazole and treated with DMSO or the USP9X inhibitor WP1130 as indicated.


A Immunoblot analysis of WT , Xiap - / - , Mcl1 - / - or cIap2 - / - mouse embryonic fibroblasts that were lentivirally infected with shRNA constructs directed against a non - relevant mRNA ( Ctrl ) or against Usp9X mRNA and treated with taxol as specified .

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A Immunoblot analysis of WT, Xiap-/-, Mcl1-/- or cIap2-/- mouse embryonic fibroblasts that were lentivirally infected with shRNA constructs directed against a non-relevant mRNA (Ctrl) or against Usp9X mRNA and treated with taxol as specified.


B Two - dimensional cell - cycle analysis ( BrdU / PI ) of cells described in ( A ) . Sub G1 and G2 / M fractions of cells were quantified and averaged with two additional , independent experiments ( n = 3 , ± SD ; WT : * * , P = 0 . 0016 ; * * * , P = 0 . 0004 ; Mcl1 - / - : * * * , P = 0 . 0003 ; * * , P = 0 . 0017 ; cIap2 - / - : * * , P = 0 . 0043 , Student ' s t test ) . Black bars exemplify shRNA Ctrl , white bars shRNA Usp9X samples .

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B Two-dimensional cell-cycle analysis (BrdU/PI) of cells described in (A). Sub G1 and G2/M fractions of cells were quantified and averaged with two additional, independent experiments (n=3, ± SD; WT: **, P = 0.0016; ***, P = 0.0004; Mcl1-/-: ***, P = 0.0003; **, P = 0.0017; cIap2-/-: **, P = 0.0043, Student's t test). Black bars exemplify shRNA Ctrl, white bars shRNA Usp9X samples.


C Immunoblot analysis of HeLa cells transfected with a FLAG - tagged USP9X expression construct or empty vector ( EV ) and treated with taxol for the indicated times .

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C Immunoblot analysis of HeLa cells transfected with a FLAG-tagged USP9X expression construct or empty vector (EV) and treated with taxol for the indicated times.


D Immunoblot analysis of HeLa cells that were transfected with a FLAG - tagged XIAP expression construct or empty vector and treated as in ( C ) .

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D Immunoblot analysis of HeLa cells that were transfected with a FLAG-tagged XIAP expression construct or empty vector and treated as in (C).


E Two dimensional cell - cycle analysis ( BrdU / PI ) of cells described in ( C ) and ( D ) . Apoptotic indices represent ratios of sub G1 to G1 / S cells and are shown for analyses at the indicated timepoints . * * , P = 0 . 0020 ; * * * , P = 0 . 0004 ; Student ' s t test .

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E Two dimensional cell-cycle analysis (BrdU/PI) of cells described in (C) and (D). Apoptotic indices represent ratios of sub G1 to G1/S cells and are shown for analyses at the indicated timepoints. **, P = 0.0020; ***, P = 0.0004; Student's t test.


F Immunoblot analysis of HeLa cells that were transfected with siRNA oligonucleotides directed against a non - relevant mRNA ( Ctrl ) or against Xiap mRNA and treated with taxol as specified .

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F Immunoblot analysis of HeLa cells that were transfected with siRNA oligonucleotides directed against a non-relevant mRNA (Ctrl) or against Xiap mRNA and treated with taxol as specified.


A Immunoblot analysis of sorted Eμ - Myc lymphoma cells that were retrovirally transduced with the indicated IRES - GFP shRNA constructs .

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A Immunoblot analysis of sorted Eμ-Myc lymphoma cells that were retrovirally transduced with the indicated IRES-GFP shRNA constructs.


B Kaplan Meier survival curves of mice that were injected intravenously with syngeneic Eμ - Myc lymphoma cells transduced with the indicated shRNA constructs as described in ( A ) . Prior to injection , cells were sorted for viability ( propidium iodide negative ) and GFP positivity ( sh _ Ctrl , black , n = 6 ; sh _ USP9X , gray , n = 5 ) . * P = 0 . 0293 ; Mantel - Cox test .

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B Kaplan Meier survival curves of mice that were injected intravenously with syngeneic Eμ-Myc lymphoma cells transduced with the indicated shRNA constructs as described in (A). Prior to injection, cells were sorted for viability (propidium iodide negative) and GFP positivity (sh_Ctrl, black, n = 6; sh_USP9X, gray, n = 5). *P = 0.0293; Mantel-Cox test.


C Kaplan - Meier survival curves of mice that were injected with syngeneic Eμ - Myc lymphoma cells transduced with the indicated shRNA constructs as described in ( A ) and treated as in ( B ) ( sh _ Ctrl , black , n = 6 ; sh _ XIAP , gray , n = 6 ) . * * , P = 0 . 0020 ; Mantel - Cox test .

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C Kaplan-Meier survival curves of mice that were injected with syngeneic Eμ-Myc lymphoma cells transduced with the indicated shRNA constructs as described in (A) and treated as in (B) (sh_Ctrl, black, n = 6; sh_XIAP, gray, n = 6). **, P = 0.0020; Mantel-Cox test.


D Histopathology of representative lymph nodes derived from mice shown in ( B ) to visualize histomorphology by haematoxylin / eosin ( H & amp ; amp ; E ) , B - cell origin ( B220 ) , and spontaneous apoptosis ( cleaved Caspase 3 ) in situ . Scale bars denote 50 μm and 20 μm for insets .

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D Histopathology of representative lymph nodes derived from mice shown in (B) to visualize histomorphology by haematoxylin/eosin (H&amp;E), B-cell origin (B220), and spontaneous apoptosis (cleaved Caspase 3) in situ. Scale bars denote 50 μm and 20 μm for insets.


E Histopathology of representative lymph nodes derived from mice shown in ( C ) which were stained as described in ( D ) .

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E Histopathology of representative lymph nodes derived from mice shown in (C) which were stained as described in (D).


F Mean tumor burden in mice injected with Eμ - Myc lymphoma cells modified as in ( B ) and ( C ) , 15 days after injection , assessed by PET imaging ( 4 animals per group ) . * * , P = 0 . 0025 ; * * * , P = 0 . 0003 , student ' s t -

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F Mean tumor burden in mice injected with Eμ-Myc lymphoma cells modified as in (B) and (C), 15 days after injection, assessed by PET imaging (4 animals per group). **, P = 0.0025; ***, P = 0.0003, student's t-test


G Representative PET images of animals , injected with Eμ - Myc lymphoma cells modified as in ( B ) and ( C ) , 15 days after injection ( pre treatment ) and three days following intraperitoneal vincristine administration ( post treatment ) . Bone marrow infiltration , representative for systemic lymphoma manifestation , is indicated by a blue asterisk .

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G Representative PET images of animals, injected with Eμ-Myc lymphoma cells modified as in (B) and (C), 15 days after injection (pre treatment) and three days following intraperitoneal vincristine administration (post treatment). Bone marrow infiltration, representative for systemic lymphoma manifestation, is indicated by a blue asterisk.


H Mean change in standardized 18FDG uptake of ROI between pre and post treatment imaging corrected for initial tumor burden ( sh _ Ctrl , black , n = 6 ; sh _ Usp9X , white , n = 8 ; sh _ Xiap , grey , n = 8 ) . Within the sh _ Usp9X and sh _ Xiap groups all animals lived up to control imaging , while two of eight animals of the sh _ ctrl group had to be sacrificed prematurely because they had reached the predefined criteria of maximum tumor burden . These animals had to be excluded from final analysis , suggesting an underestimation of the actual effect . Data was obtained from two independent experiments . * * * * , P < 0 . 0001 ; * , P = 0 . 0167 , student ' s t -

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H Mean change in standardized 18FDG uptake of ROI between pre and post treatment imaging corrected for initial tumor burden (sh_Ctrl, black, n=6; sh_Usp9X, white, n=8; sh_Xiap, grey, n=8). Within the sh_Usp9X and sh_Xiap groups all animals lived up to control imaging, while two of eight animals of the sh_ctrl group had to be sacrificed prematurely because they had reached the predefined criteria of maximum tumor burden. These animals had to be excluded from final analysis, suggesting an underestimation of the actual effect. Data was obtained from two independent experiments. ****, P < 0.0001; *, P = 0.0167, student's t-test


A Immunoblot analyses of different human DLBCL lines using antibodies to the indicated proteins .

A Immunoblot analyses of different human DLBCL lines using antibodies to the indicated proteins.


B FACS analysis ( propidium iodide ( PI ) ) of USP9X high and low expressing DLBCL cells shown in ( A ) to determine cell survival . Cells were treated with taxol and collected at the specified time points . PI negative cells are indicated relative to time point 0 ( n = 3 ± SD ) .

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B FACS analysis (propidium iodide (PI)) of USP9X high and low expressing DLBCL cells shown in (A) to determine cell survival. Cells were treated with taxol and collected at the specified time points. PI negative cells are indicated relative to time point 0 (n=3 ± SD).


C Immunoblot analyses of the indicated DLBCL cell lines that were lentivirally transduced with IRES - GFP shRNA constructs against USP9X or a non - relevant mRNA , FACS sorted for GFP + PI - cells and exposed to taxol for the indicated periods of time . The quantification of cleaved caspase 3 ( CC3 ) at the 24 hr post taxol treatment timepoint from four independent experiments is quantified in the graph on the right side and normalized to control shRNA . * * , P = 0 . 0099 , student ' s t -

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C Immunoblot analyses of the indicated DLBCL cell lines that were lentivirally transduced with IRES-GFP shRNA constructs against USP9X or a non-relevant mRNA, FACS sorted for GFP+ PI- cells and exposed to taxol for the indicated periods of time. The quantification of cleaved caspase 3 (CC3) at the 24 hr post taxol treatment timepoint from four independent experiments is quantified in the graph on the right side and normalized to control shRNA. **, P = 0.0099, student's t-test


D Immunoblot analyses of the indicated DLBCL cell lines that were exposed to taxol for the indicated periods of time . Three hours before collecting , WP1130 at a concentration of 5μM or DMSO was added as specified .

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D Immunoblot analyses of the indicated DLBCL cell lines that were exposed to taxol for the indicated periods of time. Three hours before collecting, WP1130 at a concentration of 5μM or DMSO was added as specified.


E FACS analysis ( propidium iodide ( PI ) uptake ) of DLBCL cell lines treated with taxol and / or the SMAC mimetic BV6 as indicated . Results displayed are from three independent experiments ( n = 3 ± SD ) . * * * , P = 0 . 00085 ; * * * , P = 0 . 00529 ; Student ' s t test .

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E FACS analysis (propidium iodide (PI) uptake) of DLBCL cell lines treated with taxol and/or the SMAC mimetic BV6 as indicated. Results displayed are from three independent experiments (n=3 ± SD). ***, P = 0.00085; ***, P = 0.00529; Student's t test.


F Collective assessment of USP9X and XIAP expression determined by immunohistochemistry in tissue microarrays ( TMA ) derived from a patient cohort representing 121 evaluable human CD20 + aggressive B - cell lymphomas . High USP9X expression was identified in 37 cases ( 31 % ) and significantly correlated with high XIAP expression levels ( * * , P = 0 . 004 ; Student ' s t test ) .

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F Collective assessment of USP9X and XIAP expression determined by immunohistochemistry in tissue microarrays (TMA) derived from a patient cohort representing 121 evaluable human CD20+ aggressive B-cell lymphomas. High USP9X expression was identified in 37 cases (31 %) and significantly correlated with high XIAP expression levels (**, P = 0.004; Student's t test).


G Expression data derived from ( E ) was correlated with clinical follow - up . Within a cohort of 58 patients receiving vincristine - containing chemotherapy ( CHOP regimen : cyclophosphamide , doxorubicin , vincristine , prednisone ) without the addition of the anti - CD20 antibody rituximab ( left panel ) overexpression of USP9X and XIAP ( n = 14 , straight line ) was associated with a significantly shortened event - free survival ( EFS ) as compared to all remaining patients ( n = 44 , dotted line ) ( * , P = 0 . 050 ; log rank test ) . Within a cohort of 63 patients receiving the CHOP regimen with addition of the anti - CD20 antibody rituximab overexpression of USP9X and XIAP ( n = 14 , straight line ) was not associated with a shortened event - free survival ( EFS ) as compared to all remaining patients ( n = 49 , dotted line ) ( P = 0 . 454 ) .

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G Expression data derived from (E) was correlated with clinical follow-up. Within a cohort of 58 patients receiving vincristine-containing chemotherapy (CHOP regimen: cyclophosphamide, doxorubicin, vincristine, prednisone) without the addition of the anti-CD20 antibody rituximab (left panel) overexpression of USP9X and XIAP (n = 14, straight line) was associated with a significantly shortened event-free survival (EFS) as compared to all remaining patients (n = 44, dotted line) (*, P = 0.050; log rank test). Within a cohort of 63 patients receiving the CHOP regimen with addition of the anti-CD20 antibody rituximab overexpression of USP9X and XIAP (n = 14, straight line) was not associated with a shortened event-free survival (EFS) as compared to all remaining patients (n = 49, dotted line) (P = 0.454).


molecular detection of nCoV - 2019 in seven patients during two times of sampling . Patient information can be found in Extended Data Table 1 and 2 . Details on detection method can be found in material and methods . BALF , bronchoalveolar lavage fluid ; OS , oral swab ; AS , anal swab .

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molecular detection of nCoV-2019 in seven patients during two times of sampling. Patient information can be found in Extended Data Table 1 and 2. Details on detection method can be found in material and methods. BALF, bronchoalveolar lavage fluid; OS, oral swab; AS, anal swab.


dynamics of nCoV - 2019 antibodies in one patient who showed sign of disease on 2019 . 12 . 23 ( ICU - 06 ) . For b and c , cut - off was set up as 0 . 2 for IgM test and 0 . 3 for IgG test , according to healthy controls .

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dynamics of nCoV-2019 antibodies in one patient who showed sign of disease on 2019.12.23 (ICU-06). For b and c, cut-off was set up as 0.2 for IgM test and 0.3 for IgG test, according to healthy controls.


serological test of nCoV - 2019 antibodies in five patients ( more information can be found in Extended Data Table 2 ) . Star indicates data collected from patient ICU - 06 on 2020 . 01 . 10 . For b and c , cut - off was set up as 0 . 2 for IgM test and 0 . 3 for IgG test , according to healthy controls .

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serological test of nCoV-2019 antibodies in five patients (more information can be found in Extended Data Table 2). Star indicates data collected from patient ICU-06 on 2020.01.10. For b and c, cut-off was set up as 0.2 for IgM test and 0.3 for IgG test, according to healthy controls.


viral particles in the ultrathin sections under electron microscope at 200 kV , sample from viral infected Vero E6

viral particles in the ultrathin sections under electron microscope at 200 kV, sample from viral infected Vero E6 cells


usage . Determination of virus infectivity in HeLa cells with or without the expression of ACE2 . h , human ; b , Rhinolophus sinicus bat ; c , civet ; s , swine ( pig ) ; m , mouse . ACE2 protein ( green ) , viral protein ( red ) and nuclei ( blue ) was shown . Scale bar = 10 um .

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usage.Determination of virus infectivity in HeLa cells with or without the expression of ACE2. h, human; b, Rhinolophus sinicus bat; c, civet; s, swine (pig); m, mouse. ACE2 protein (green), viral protein (red) and nuclei (blue) was shown. Scale bar=10 um.


A . RT - PCR analysis of the expression of β - actin , Trpv1 , Trpv2 , Trpv3 and Trpv4 using mouse differentiated brown adipocytes after 29 ( upper ) and 35 ( lower ) thermal cycles with ( RT ( + ) ) and without ( RT ( - ) ) reverse transcription ( RT ) . Control ( Ct . ) lanes indicate the results with each plasmid DNA as a template .

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A. RT-PCR analysis of the expression of β-actin, Trpv1, Trpv2, Trpv3 and Trpv4 using mouse differentiated brown adipocytes after 29 (upper) and 35 (lower) thermal cycles with (RT (+)) and without (RT (-)) reverse transcription (RT). Control (Ct.) lanes indicate the results with each plasmid DNA as a template.


B . Results of real - time RT - PCR analysis of Trpv1 , Trpv2 , Trpv3 and Trpv4 expression using mouse differentiated brown adipocytes . Expression levels of mRNA were normalized to that of the ribosomal protein gene ( 36B4 ) , a housekeeping gene un - affected by adipogenesis . Data are presented as mean ± SEM , n = 6 .

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B. Results of real-time RT-PCR analysis of Trpv1, Trpv2, Trpv3 and Trpv4 expression using mouse differentiated brown adipocytes. Expression levels of mRNA were normalized to that of the ribosomal protein gene (36B4), a housekeeping gene un-affected by adipogenesis. Data are presented as mean ± SEM, n = 6.


C . Results of real - time PCR analysis of Trpv1 , Trpv2 , Trpv3 and Trpv4 expression using mouse interscapular brown adipose tissue ( iBAT ) . mRNA expression levels were normalized to that of 36B4 . Data are presented as mean ± SEM , n = 5 .

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C. Results of real-time PCR analysis of Trpv1, Trpv2, Trpv3 and Trpv4 expression using mouse interscapular brown adipose tissue (iBAT). mRNA expression levels were normalized to that of 36B4. Data are presented as mean ± SEM, n = 5.


D . Western blot results of TRPV2 and tubulin from WT and TRPV2KO brown adipocytes . Upper bands in the TRPV2 blots likely indicate glycosylated forms .

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D. Western blot results of TRPV2 and tubulin from WT and TRPV2KO brown adipocytes. Upper bands in the TRPV2 blots likely indicate glycosylated forms.


E . Averaged traces of [ Ca2 + ] i changes in response to 500 µmol / L 2APB in differentiated brown adipocytes from WT ( black ) and TRPV2KO ( red ) mice . One µmol / L NE was used to confirm differentiation . Five µmol / L ionomycin was used to confirm cell viability . Ratio values correspond to the real [ Ca2 + ] i of differentiated mousebrown adipocytes . Data are presented as mean ± SEM , n = 149 of WT cells , and n = 112 of TRPV2KO brown adipocytes ; * * P < 0 . 01 . Unpaired Student ' s t - test .

O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

E. Averaged traces of [Ca2+]i changes in response to 500 µmol/L 2APB in differentiated brown adipocytes from WT (black) and TRPV2KO (red) mice. One µmol/L NE was used to confirm differentiation. Five µmol/L ionomycin was used to confirm cell viability. Ratio values correspond to the real [Ca2+]i of differentiated mousebrown adipocytes. Data are presented as mean ± SEM, n = 149 of WT cells, and n = 112 of TRPV2KO brown adipocytes; ** P < 0.01. Unpaired Student's t-test.


F . A representative trace of whole - cell current activated by 3 mmol / L of 2APB in the presence or absence of 10 µmol / L SKF in mouse differentiated brown adipocyte . The left inset indicates a voltage ramp - pulse protocol . The right inset indicates the current - voltage curves of basal current ( a , black ) , current in the presence of 2APB + SKF ( b , blue ) and current in the presence of 2APB alone ( c , red ) at the time points of a , b and c , respectively . G . Comparison of the mean densities of basal currents ( Base , black ) , currents in the presence of 2APB alone ( red ) and currents in the presence of 2APB + SKF ( blue ) at - 60 mV and + 100 mV in mouse differentiated brown adipocytes . Data are presented as mean ± SEM , n = 10 ; * * P < 0 . 01 vs . Base ; # # P < 0 . 01 vs . 2APB alone . One - way ANOVA followed by 2 - tailed t - test with Bonferroni correction .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. A representative trace of whole-cell current activated by 3 mmol/L of 2APB in the presence or absence of 10 µmol/L SKF in mouse differentiated brown adipocyte. The left inset indicates a voltage ramp-pulse protocol. The right inset indicates the current-voltage curves of basal current (a, black), current in the presence of 2APB+SKF (b, blue) and current in the presence of 2APB alone (c, red) at the time points of a, b and c, respectively.G. Comparison of the mean densities of basal currents (Base, black), currents in the presence of 2APB alone (red) and currents in the presence of 2APB+SKF (blue) at -60 mV and +100 mV in mouse differentiated brown adipocytes. Data are presented as mean ± SEM, n = 10; ** P < 0.01 vs. Base; ##P < 0.01 vs. 2APB alone. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.


A . Results of real - time RT - PCR analysis of Trpv1 , Trpv2 , Trpv3 and Trpv4 expression in mouse pre - adipocytes and differentiated brown adipocytes . Expression levels of mRNA were normalized to those of 36B4 . Data are presented as mean ± SEM , n = 6 . * P < 0 . 05 vs . pre - adipocytes . Unpaired Student ' s t - test .

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A. Results of real-time RT-PCR analysis of Trpv1, Trpv2, Trpv3 and Trpv4 expression in mouse pre-adipocytes and differentiated brown adipocytes. Expression levels of mRNA were normalized to those of 36B4. Data are presented as mean ± SEM, n = 6. * P < 0.05 vs. pre-adipocytes. Unpaired Student's t-test.


B . The number of 6 day - differentiated mousebrown adipocytes treated with TRPV2 agonists , 100 µmol / L 2APB or 10 µmol / L LPC . Mean ± SEM , n = 6 , * P < 0 . 05 and * * P < 0 . 01 vs . dimethyl sulfoxide ( DMSO ) group . One - way ANOVA followed by 2 - tailed t - test with Bonferroni correction .

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B. The number of 6 day-differentiated mousebrown adipocytes treated with TRPV2 agonists, 100 µmol/L 2APB or 10 µmol/L LPC. Mean ± SEM, n = 6, * P < 0.05 and ** P < 0.01 vs. dimethyl sulfoxide (DMSO) group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.


C and D . Comparison of the numbers of 6 day - differentiated mouse brown adipocytes ( C ) and triglyceride levels ( D ) in the cells from WT and TRPV2KO mice with different differentiation media . 1 / 10 suppl . indicates differentiation medium ten - time - diluted with DMEM . Mean ± SEM , n = 8 , * * P < 0 . 01 vs . control group , # # P < 0 . 01 vs . WT group . One - way ANOVA followed by 2 - tailed t - test with Bonferroni correction .

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C and D. Comparison of the numbers of 6 day-differentiated mouse brown adipocytes (C) and triglyceride levels (D) in the cells from WT and TRPV2KO mice with different differentiation media. 1/10 suppl. indicates differentiation medium ten-time-diluted with DMEM. Mean ± SEM, n = 8, ** P < 0.01 vs. control group, ##P < 0.01 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.


A . Basal mRNA expression of Ucp1 , peroxisome proliferator - activated receptor gamma coactivator 1 - alpha ( Pgc1α ) , peroxisome proliferator - activated receptorγ ( Pparγ ) and β3 - adrenergic receptor 3 ( Adrb3 ) in the differentiated brown adipocytes from WT and TRPV2KO mice . Data are presented as mean ± SEM , n = 5 ; * * P < 0 . 01 vs . WT . Unpaired Student ' s t - test .

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A. Basal mRNA expression of Ucp1, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc1α), peroxisome proliferator-activated receptorγ(Pparγ) and β3-adrenergic receptor 3 (Adrb3) in the differentiated brown adipocytes from WT and TRPV2KO mice. Data are presented as mean ± SEM, n = 5; ** P < 0.01 vs. WT. Unpaired Student's t-test.


B and C . Changes in Ucp1 ( B ) and Pgc1α ( C ) mRNA expression in the differentiated brown adipocytes from WT and TRPV2KO mice with and without 10 µmol / L isoproterenol ( ISO ) for 4 h . Data are presented as mean ± SEM , n = 5 ; * * P < 0 . 01 vs . DMSO group ; # P < 0 . 05 ; # # P < 0 . 01 vs . WT group . One - way ANOVA followed by 2 - tailed t - test with Bonferroni correction .

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B and C. Changes in Ucp1 (B) and Pgc1α(C) mRNA expression in the differentiated brown adipocytes from WT and TRPV2KO mice with and without 10 µmol/L isoproterenol (ISO) for 4 h. Data are presented as mean ± SEM, n = 5; ** P < 0.01 vs. DMSO group; # P < 0.05; ## P < 0.01 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.


D and E . Changes in Ucp1 mRNA expression ( D ) and non - esterified fatty acid ( NEFA ) release ( E ) in the differentiated brown adipocytes from WT and TRPV2KO mice with or without 10 µmol / L forskolin for 4 h . Data are presented as mean ± SEM , n = 6 ; * P < 0 . 05 ; * * P < 0 . 01 vs . DMSO group ; # # P < 0 . 01 vs . WT group . One - way ANOVA followed by 2 - tailed t - test with Bonferroni correction .

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D and E. Changes in Ucp1 mRNA expression (D) and non-esterified fatty acid (NEFA) release (E) in the differentiated brown adipocytes from WT and TRPV2KO mice with or without 10 µmol/L forskolin for 4 h. Data are presented as mean ± SEM, n = 6; * P < 0.05; ** P < 0.01 vs. DMSO group; ## P < 0.01 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.


F and G . Changes in Ucp1 mRNA ( F ) and Pgc1α mRNA ( G ) in the differentiated brown adipocytes treated with 10 µmol / L ISO alone or 10 µmol / L ISO plus 10 µmol / L BAPTA - AM for 4 h . Data are presented as mean ± SEM , n = 6 ; * P < 0 . 05 ; * * P < 0 . 01 vs . DMSO group ; # # P < 0 . 01 vs . ISO group . One - way ANOVA followed by 2 - tailed t - test with Bonferroni correction .

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F and G. Changes in Ucp1 mRNA (F) and Pgc1α mRNA (G) in the differentiated brown adipocytes treated with 10 µmol/L ISO alone or 10 µmol/L ISO plus 10 µmol/L BAPTA-AM for 4 h. Data are presented as mean ± SEM, n = 6; * P < 0.05; ** P < 0.01 vs. DMSO group; ## P < 0.01 vs. ISO group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.


A . Body weight changes from 3 - weeks to 8 - weeks of age between the two genotypes ( n = 11 ) . Data are presented as mean ± SEM ; * P < 0 . 05 ; * * P < 0 . 01 vs . WT group . Unpaired Student ' s t - test .

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A. Body weight changes from 3-weeks to 8-weeks of age between the two genotypes (n = 11). Data are presented as mean ± SEM; * P < 0.05; ** P < 0.01 vs. WT group. Unpaired Student's t-test.


B and C . Changes in food intake ( B ) and water intake ( C ) from 3 - weeks to 8 - weeks of ages between WT and TRPV2KO mice ( n = 6 ) .

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B and C. Changes in food intake (B) and water intake (C) from 3-weeks to 8-weeks of ages between WT and TRPV2KO mice (n = 6).


D . Weights of tissues related to energy metabolism normalized to their body weights in WT and TRPV2KO mice ( n = 15 ) .

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D. Weights of tissues related to energy metabolism normalized to their body weights in WT and TRPV2KO mice (n = 15).


E . The expression levels of mRNA related to energy metabolism in iBAT from WT and TRPV2KO mice . mRNA expression levels of Ucp1 , Pgc1a , Ppary , Adrb3 , cytochrome c oxidase subunit 4 isoform 1 ( Cox4i1 ) , PR domain containing 16 ( Prdm16 ) , Ucp2 , lipoprotein lipase ( Lpl ) and cluster of differentiation 36 ( Cd36 ) were examined and normalized to the levels of 36B4 . Data are presented as mean ± SEM , n = 15 ; * P < 0 . 05 vs . WT group ; * * P < 0 . 01 vs . WT group . Unpaired Student ' s t - test .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 expression levels of mRNA related to energy metabolism in iBAT from WT and TRPV2KO mice. mRNA expression levels of Ucp1, Pgc1a, Ppary, Adrb3, cytochrome c oxidase subunit 4 isoform 1 (Cox4i1), PR domain containing 16 (Prdm16), Ucp2, lipoprotein lipase (Lpl) and cluster of differentiation 36 (Cd36) were examined and normalized to the levels of 36B4. Data are presented as mean ± SEM, n = 15; * P < 0.05 vs. WT group; ** P < 0.01 vs. WT group. Unpaired Student's t-test.


A . Representative morphological results of iBAT by hematoxylin and eosin staining from 8 - week WT and TRPV2KO mice . Scale bars indicate 100 μm . B . Comparison of lipid droplet size histograms from 8 - week - old WT ( black ) and TRPV2KO ( red ) iBAT with hematoxylin and eosin staining . Mean values are 9 . 5 ± 0 . 2 µm in 8 - week - old WT adipocytes , and 14 . 7 ± 0 . 2 µm in 8 - week - old TRPV2KO adipocytes ( P < 0 . 01 ) . 1000 to 1400 droplets were counted . Data are presented as mean ± SEM , n = 4 . C . Comparison of adipocyte size histograms from 8 - week - old WT ( black ) and TRPV2KO ( red ) iBAT with hematoxylin and eosin staining . 800 to 1000 cells were counted . Data are presented as mean ± SEM , n = 4 . D . Mean diameters of adipocytes in 8 - week - old WT mice ( black ) and TRPV2KO mice ( red ) . Mean values are 15 . 8 ± 0 . 4 µm in 8 - week - old WT adipocytes , and 19 . 8 ± 0 . 3 µm in 8 - week - old TRPV2KO adipocytes . Data are presented as mean ± SEM , n = 4 ; * P < 0 . 05 vs . WT group . Unpaired Student ' s t - test .

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A. Representative morphological results of iBAT by hematoxylin and eosin staining from 8-week WT and TRPV2KO mice. Scale bars indicate 100 μm.B. Comparison of lipid droplet size histograms from 8-week-old WT (black) and TRPV2KO (red) iBAT with hematoxylin and eosin staining. Mean values are 9.5 ± 0.2 µm in 8-week-old WT adipocytes, and 14.7 ± 0.2 µm in 8-week-old TRPV2KO adipocytes (P < 0.01). 1000 to 1400 droplets were counted. Data are presented as mean ± SEM, n = 4.C. Comparison of adipocyte size histograms from 8-week-old WT (black) and TRPV2KO (red) iBAT with hematoxylin and eosin staining. 800 to 1000 cells were counted. Data are presented as mean ± SEM, n = 4.D. Mean diameters of adipocytes in 8-week-old WT mice (black) and TRPV2KO mice (red). Mean values are 15.8 ± 0.4 µm in 8-week-old WT adipocytes, and 19.8 ± 0.3 µm in 8-week-old TRPV2KO adipocytes. Data are presented as mean ± SEM, n = 4; * P < 0.05 vs. WT group. Unpaired Student's t-test.


A , B , C and D . Changes in mRNA expression of Ucp1 ( A ) , Pgc1α ( B ) , Pparγ ( C ) and Trpv2 ( D ) in iBAT of WT and TRPV2KO mice before ( 25oC ) , after 1 day and 3 days cold ( 4oC ) exposure . Mean ± SEM , n = 5 - 8 ; * P < 0 . 05 ; * * P < 0 . 01 vs . 25oC group ; # P < 0 . 05 vs . WT group . One - way ANOVA followed by 2 - tailed t - test with Bonferroni correction .

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A, B, C and D. Changes in mRNA expression of Ucp1 (A), Pgc1α (B), Pparγ (C) and Trpv2 (D) in iBAT of WT and TRPV2KO mice before (25oC), after 1 day and 3 days cold (4oC) exposure. Mean ± SEM, n = 5 - 8; * P < 0.05; ** P < 0.01 vs. 25oC group; # P < 0.05 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.


E and F . Western blot results of UCP1 protein in iBAT from WT and TRPV2KO mice before ( 25oC ) and after 3 days cold ( 4oC ) exposure ( E ) . Comparison of UCP1 protein levels before and after cold exposure in iBAT from WT and TRPV2KO mice ( F ) . Mean ± SEM , n = 6 ; * P < 0 . 05 vs . 25oC group ; # P < 0 . 05 vs . WT group . One - way ANOVA followed by 2 - tailed t - test with Bonferroni correction .

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E and F. Western blot results of UCP1 protein in iBAT from WT and TRPV2KO mice before (25oC) and after 3 days cold (4oC) exposure (E). Comparison of UCP1 protein levels before and after cold exposure in iBAT from WT and TRPV2KO mice (F). Mean ± SEM, n = 6; * P < 0.05 vs. 25oC group; # P < 0.05 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.


G and H . Averages of core body temperatures ( G ) and activities ( H ) of WT and TRPV2KO mice with or without cold ( 4oC ) exposure . Data are presented as mean ± SEM , n = 5 ; * P < 0 . 05 vs . WT group . Unpaired Student ' s t - test .

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G and H. Averages of core body temperatures (G) and activities (H) of WT and TRPV2KO mice with or without cold (4oC) exposure. Data are presented as mean ± SEM, n = 5; * P < 0.05 vs. WT group. Unpaired Student's t-test.


A , B and C . Changes in mRNA expression of Ucp1 ( A ) , Pgc1α ( B ) and Pparγ ( C ) from WT and TRPV2KO iBAT 4 h after intraperitoneal administration ( i . p . , arrows ) of saline or a selective β3 - adrenergic receptor agonist , BRL37344 ( 600 µg / kg body weight ) . Mean ± SEM , n = 5 - 7 ; * P < 0 . 05 vs . saline group ; # P < 0 . 01 vs . WT BRL37344 administration group . One - way ANOVA followed by 2 - tailed t - test with Bonferroni correction .

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A, B and C. Changes in mRNA expression of Ucp1 (A), Pgc1α (B) and Pparγ(C) from WT and TRPV2KO iBAT 4 h after intraperitoneal administration (i.p., arrows) of saline or a selective β3-adrenergic receptor agonist, BRL37344 (600 µg/kg body weight). Mean ± SEM, n = 5 - 7; * P < 0.05 vs. saline group; # P < 0.01 vs. WT BRL37344 administration group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.


D and E . Average traces of changes in iBAT temperature ( D ) or rectal body temperature ( E ) from WT and TRPV2KO mice after BRL37344 ( 600 µg / kg body weight ) administration ( i . p . , arrows ) . Mean ± SEM , n = 5 . * P < 0 . 05 vs . WT group . Unpaired Student ' s t - test .

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D and E. Average traces of changes in iBAT temperature (D) or rectal body temperature (E) from WT and TRPV2KO mice after BRL37344 (600 µg/kg body weight) administration (i.p., arrows). Mean ± SEM, n = 5. * P < 0.05 vs. WT group. Unpaired Student's t-test.


F and G . iBAT NE turnover rates ( F ) and rate constants k ( G ) in WT and TRPV2KO mice upon cold exposure ( 4oC ) for 4 h . Data are presented as mean ± SEM , n = 8 .

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F and G. iBAT NE turnover rates (F) and rate constants k (G) in WT and TRPV2KO mice upon cold exposure (4oC) for 4 h. Data are presented as mean ± SEM, n = 8.


A . Body weight changes between WT and TRPV2KO mice treated with high fat diet ( HFD ) for continuous 8 weeks from 13 - week - old .

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A. Body weight changes between WT and TRPV2KO mice treated with high fat diet (HFD) for continuous 8 weeks from 13-week-old.


B and C . Blood glucose level ( B ) and plasma insulin level ( C ) of WT and TRPV2KO mice after 8 - week HFD treatment .

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B and C. Blood glucose level (B) and plasma insulin level (C) of WT and TRPV2KO mice after 8-week HFD treatment.


D . Weights of tissues related to energy metabolism in WT and TRPV2KO mice after 8 - week HFD treatment .

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D. Weights of tissues related to energy metabolism in WT and TRPV2KO mice after 8-week HFD treatment.


E . mRNA expression of Ucp1 and Pgc1α in iBAT from WT and TRPV2KO mice after 8 - week HFD treatment . All data are presented as mean ± SEM , WT mice ( n = 6 ) , TRPV2KO mice ( n = 8 ) ; * P < 0 . 05 ; * * P < 0 . 01 vs . WT group . Unpaired Student ' s t - test .

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E. mRNA expression of Ucp1 and Pgc1α in iBAT from WT and TRPV2KO mice after 8-week HFD treatment. All data are presented as mean ± SEM, WT mice (n = 6), TRPV2KO mice (n = 8); * P < 0.05; ** P < 0.01 vs. WT group. Unpaired Student's t-test.


B Analysis of anti - Bax and anti - Bak antibody specificity using whole cell lysates from HCT116 wild - type , Bax KO , Bak KO and Bax / Bak DKO . After detection of Bax the membrane was redecorated with anti - Bak antibodies ( Bax / Bak ) . Actin is used as loading control .

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B Analysis of anti-Bax and anti-Bak antibody specificity using whole cell lysates from HCT116 wild-type, Bax KO, Bak KO and Bax/Bak DKO. After detection of Bax the membrane was redecorated with anti-Bak antibodies (Bax/Bak). Actin is used as loading control.


C Western blot analysis of Bak in the heavy membrane ( HM ) fraction of human heart , cerebral cortex ( CNS GM ) , kidney , brain white matter ( CNS WM ) , lung , liver , and skeletal muscle tissue . The cytosol fraction obtained from CNS cortex ( C ) , LDH , and Tom20 serve as fractionation and loading controls . n = 3 .

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C Western blot analysis of Bak in the heavy membrane (HM) fraction of human heart, cerebral cortex (CNS GM), kidney, brain white matter (CNS WM), lung, liver, and skeletal muscle tissue. The cytosol fraction obtained from CNS cortex (C), LDH, and Tom20 serve as fractionation and loading controls. n = 3.


D Presence of Bak in the cytosol of heart , cerebral cortex ( CNS GM ) , kidney , brain white matter ( CNS WM ) , lung , liver , and skeletal muscle tissue analyzed by Western blot in parallel to C . The heavy membrane heart fraction ( HM ) , LDH and Tom20 serve as fractionation and loading controls . n = 3 .

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D Presence of Bak in the cytosol of heart, cerebral cortex (CNS GM), kidney, brain white matter (CNS WM), lung, liver, and skeletal muscle tissue analyzed by Western blot in parallel to C. The heavy membrane heart fraction (HM), LDH and Tom20 serve as fractionation and loading controls. n = 3.


E Carbonate extraction of the heavy membrane protein fraction of human brain white matter ( CNS WM ) and lung tissue analyzed for the presence of Bak by Western blot . Membrane - associated protein supernatant ( S ) and membrane - integral protein pellet ( P ) are displayed . Smac is released from the mitochondrial inter membrane space ( IMS ) during carbonate extraction and CoxIV remains in the pellet . n = 2 .

O O 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-MEASURED_VAR 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-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O

E Carbonate extraction of the heavy membrane protein fraction of human brain white matter (CNS WM) and lung tissue analyzed for the presence of Bak by Western blot. Membrane-associated protein supernatant (S) and membrane-integral protein pellet (P) are displayed. Smac is released from the mitochondrial inter membrane space (IMS) during carbonate extraction and CoxIV remains in the pellet. n = 2.


A Confocal images of HCT116 Bax / Bak DKO cells transfected with GFP - Bak in the absence ( left ) or the presence ( right ) of Bcl - xL overexpression . Scale bar 15 µm . n ≥ 10 .

O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O

A Confocal images of HCT116 Bax/Bak DKO cells transfected with GFP-Bak in the absence (left) or the presence (right) of Bcl-xL overexpression. Scale bar 15 µm. n ≥ 10.


B FLIP ( Fluorescence Loss in Photobleaching ) of mitochondrial GFP - Bak in the absence ( ● , straight line ) or the presence ( ○ , broken line ) of overexpressed Bcl - xL . Fluorescence of the neighboring cell ( in Fig . 2C ) is shown as control ( , dotted line ) . Data represent averages ± SEM from 16 ( - Bcl - xL ) and 40 ( + Bcl - xL ) ROI measurements .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O

B FLIP (Fluorescence Loss in Photobleaching) of mitochondrial GFP-Bak in the absence (●, straight line) or the presence (○, broken line) of overexpressed Bcl-xL. Fluorescence of the neighboring cell (in Fig. 2C) is shown as control (, dotted line). Data represent averages ± SEM from 16 (-Bcl-xL) and 40 (+Bcl-xL) ROI measurements.


C FLIP of GFP - Bak in the absence ( top ) or the presence ( bottom ) of overexpressed Bcl - xL diminishes GFP - Bak fluorescence in the cytosol of targeted cells ( circled ) completely after 90 s and GFP fluorescence is detected only on the mitochondria ( arrows ) . During FLIP measurements mitochondrial GFP - Bak fluorescence is monitored , while the cytosol is bleached repeatedly . Time points in seconds are displayed above the images .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

C FLIP of GFP-Bak in the absence (top) or the presence (bottom) of overexpressed Bcl-xL diminishes GFP-Bak fluorescence in the cytosol of targeted cells (circled) completely after 90 s and GFP fluorescence is detected only on the mitochondria (arrows). During FLIP measurements mitochondrial GFP-Bak fluorescence is monitored, while the cytosol is bleached repeatedly. Time points in seconds are displayed above the images.


A Western blot analysis of GFP - Bak localization in cytosol ( C ) and heavy membrane fraction ( HM ) of HCT116 Bax / Bak DKO cells with or without overexpressed wild - type Bcl - xL or Bcl - xL G138A . GAPDH and Tom20 serve as fractionation controls . n = 10 . Bak shuttling by Bcl - xL is independent of the GFP fusion ( Fig . E2a ) . B Quantification of predominant mitochondrial localization ( light grey bars ) and mixed cytosolic and mitochondrial localization ( dark grey bars ) of Bak in HCT116 Bax / Bak DKO cells expressing GFP - Bak with or without Bcl - xL overexpression . Data represent averages ± SD from 3 independent experiments with n ≥ 100 .

O O O O O O O B-MEASURED_VAR O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O 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-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O

A Western blot analysis of GFP-Bak localization in cytosol (C) and heavy membrane fraction (HM) of HCT116 Bax/Bak DKO cells with or without overexpressed wild-type Bcl-xL or Bcl-xL G138A. GAPDH and Tom20 serve as fractionation controls. n = 10. Bak shuttling by Bcl-xL is independent of the GFP fusion (Fig. E2a).B Quantification of predominant mitochondrial localization (light grey bars) and mixed cytosolic and mitochondrial localization (dark grey bars) of Bak in HCT116 Bax/Bak DKO cells expressing GFP-Bak with or without Bcl-xL overexpression. Data represent averages ± SD from 3 independent experiments with n ≥ 100.


C Retrotranslocation rates measured for wild - type Bak or Bak D83R in the presence or the absence of Bcl - 2 , Mcl - 1 , wild - type Bcl - xL or Bcl - xL G138A . Data represent averages ± SD .

O O O O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O

C Retrotranslocation rates measured for wild-type Bak or Bak D83R in the presence or the absence of Bcl-2, Mcl-1, wild-type Bcl-xL or Bcl-xL G138A. Data represent averages ± SD.


D Analysis of endogenous Bak localization in cytosol ( C ) and heavy membrane fraction ( HM ) of HeLa cells expressing degradation - prone DD - FLAG - Bcl - xL . In the absence of Shield - 1 DD - FLAG - Bcl - xL is readily degraded , resulting in unaltered Bcl - xL levels . Addition of 0 . 5 µM Shield - 1 instantly stabilizes DD - FLAG - Bcl - xL resulting in elevated Bcl - xL levels that increase cytosolic levels of endogenous Bak ( Fig . E3C , D ) . Akt1 and Tom20 serve as fractionation controls . n = 3 .

O O O O B-MEASURED_VAR 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

D Analysis of endogenous Bak localization in cytosol (C) and heavy membrane fraction (HM) of HeLa cells expressing degradation-prone DD-FLAG-Bcl-xL. In the absence of Shield-1 DD-FLAG-Bcl-xL is readily degraded, resulting in unaltered Bcl-xL levels. Addition of 0.5 µM Shield-1 instantly stabilizes DD-FLAG-Bcl-xL resulting in elevated Bcl-xL levels that increase cytosolic levels of endogenous Bak (Fig. E3C, D). Akt1 and Tom20 serve as fractionation controls. n = 3.


B Quantification of HCT116 Bax / Bak DKO cells expressing Bax , BaxTBak , BakTBax or Bak with the expressed protein being present largely cytosolic ( white bars ) , in a mixed distribution between cytosol and mitochondria ( grey bars ) or largely mitochondrial ( black bars ) . Data represent averages ± SEM from 7 independent experiments with n ≥ 100 cells .

O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O B-MEASURED_VAR O B-CONTROLLED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O 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 Quantification of HCT116 Bax/Bak DKO cells expressing Bax, BaxTBak, BakTBax or Bak with the expressed protein being present largely cytosolic (white bars), in a mixed distribution between cytosol and mitochondria (grey bars) or largely mitochondrial (black bars). Data represent averages ± SEM from 7 independent experiments with n ≥ 100 cells.


C Confocal images of HCT116 Bax / Bak DKO cells expressing Bax , BaxTBak , BakTBax or Bak . The GFP / YFP fluorescence of the expressed protein variants is depicted in the top panels and in green in the merged image on the bottom . The mitochondria were stained by MitoTracker far red depicted in red in the merged images ( bottom row ) . Scale 10 µm . n ≥ 5 .

O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O B-MEASURED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Confocal images of HCT116 Bax/Bak DKO cells expressing Bax, BaxTBak, BakTBax or Bak. The GFP/YFP fluorescence of the expressed protein variants is depicted in the top panels and in green in the merged image on the bottom. The mitochondria were stained by MitoTracker far red depicted in red in the merged images (bottom row). Scale 10 µm. n ≥ 5.


D Western blot analysis of Bax , BaxTBak , Bak and BakTBax localization expressed in HCT116 Bax / Bak DKO cells . Cytosol ( C ) and heavy membrane fraction ( HM ) of HCT116 Bax / Bak DKO cells are displayed . GAPDH and VDAC serve as fractionation controls . n = 3 .

O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O

D Western blot analysis of Bax, BaxTBak, Bak and BakTBax localization expressed in HCT116 Bax/Bak DKO cells. Cytosol (C) and heavy membrane fraction (HM) of HCT116 Bax/Bak DKO cells are displayed. GAPDH and VDAC serve as fractionation controls. n = 3.


A Caspase 3 / 7 activity measured in HCT116 Bax / Bak DKO cells overexpressing Bax , BaxTBak , Bak or BakTBax with or without Bcl - xL overexpression in the absence of apoptosis stimuli . Caspase activity is displayed in relative fluorescence units ( RFU ) . pcDNA3 . 1 - transfected cells served as control . Data represent averages ± SEM . n ≥ 3 . p - values according to One Way ANOVA are displayed . B Staurosporine ( STS , 1 µM ) - induced caspase 3 / 7 activity of Bax / Bak DKO cells overexpressing Bax , BaxTBak , Bak or BakTBax with or without Bcl - xL overexpression displayed in relative fluorescence units ( RFU ) . Data represent averages ± SEM . n ≥ 3 . p - values according to One Way ANOVA . BaxTBak activities with and without Bcl - xL expression revealed no significant difference ( n . s . ) , while in the presence of Bcl - xL overexpression BaxTBak activity is significantly higher than Bax , Bak or BakTBax activities ( p < 0 . 001 ) .

O B-MEASURED_VAR I-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O 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-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O

A Caspase 3/7 activity measured in HCT116 Bax/Bak DKO cells overexpressing Bax, BaxTBak, Bak or BakTBax with or without Bcl-xL overexpression in the absence of apoptosis stimuli. Caspase activity is displayed in relative fluorescence units (RFU). pcDNA3.1-transfected cells served as control. Data represent averages ± SEM. n ≥ 3. p-values according to One Way ANOVA are displayed.B Staurosporine (STS, 1 µM)-induced caspase 3/7 activity of Bax/Bak DKO cells overexpressing Bax, BaxTBak, Bak or BakTBax with or without Bcl-xL overexpression displayed in relative fluorescence units (RFU). Data represent averages ± SEM. n ≥ 3. p-values according to One Way ANOVA. BaxTBak activities with and without Bcl-xL expression revealed no significant difference (n.s.), while in the presence of Bcl-xL overexpression BaxTBak activity is significantly higher than Bax, Bak or BakTBax activities (p < 0.001).


C Analysis of the active Bax conformation in HCT116 Bax / Bak DKO cells expressing wild - type Bax or BaxTBak with ( dark grey bars ) or without Bcl - xL overexpression ( light grey bars ) by the monoclonal antibody 6A7 ( Sigma ) detecting the active Bax protein fold by fluorescence imaging . Cells were analyzed prior or after treatment with 1 µM STS in the presence of the pan - caspase inhibitor qVD . Data are represented as % of the expressing cell population ± SEM . n = 4 .

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C Analysis of the active Bax conformation in HCT116 Bax/Bak DKO cells expressing wild-type Bax or BaxTBak with (dark grey bars) or without Bcl-xL overexpression (light grey bars) by the monoclonal antibody 6A7 (Sigma) detecting the active Bax protein fold by fluorescence imaging. Cells were analyzed prior or after treatment with 1 µM STS in the presence of the pan-caspase inhibitor qVD. Data are represented as % of the expressing cell population ± SEM. n = 4.


D HCT116 Bax / Bak DKO cells ectopically expressing wild - type Bax or BaxTBak with ( dark grey bars ) or without Bcl - xL ( light grey bars ) were analyzed in the presence or the absence of 1 µM STS and qVD for retained mitochondrial cyt c . Data are represented as % of the expressing cell population ± SEM . n = 4 .

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D HCT116 Bax/Bak DKO cells ectopically expressing wild-type Bax or BaxTBak with (dark grey bars) or without Bcl-xL (light grey bars) were analyzed in the presence or the absence of 1 µM STS and qVD for retained mitochondrial cyt c. Data are represented as % of the expressing cell population ± SEM. n = 4.


E Flow cytometry analysis of Annexin V - staining of Bax / Bak DKO cells expressing Bax , BaxTBak , Bak or BakTBax in the absence ( red line ) or the presence of Bcl - xL overexpression ( black line ) , following STS - treatment . The percentage of gated cells is displayed in the color of the corresponding graph . Data represent averages ± SD . n = 4 .

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E Flow cytometry analysis of Annexin V-staining of Bax/Bak DKO cells expressing Bax, BaxTBak, Bak or BakTBax in the absence (red line) or the presence of Bcl-xL overexpression (black line), following STS-treatment. The percentage of gated cells is displayed in the color of the corresponding graph. Data represent averages ± SD. n = 4.


F Colony formation of Bax / Bak DKO cells transfected with pcDNA , Bax , BaxTBak , Bak or BakTBax with or without Bcl - xL overexpression . STS ( 1 µM ) was added for 24 hrs before cells were replated and colonies were stained with methylene blue typically 14 days after treatment .

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F Colony formation of Bax/Bak DKO cells transfected with pcDNA, Bax, BaxTBak, Bak or BakTBax with or without Bcl-xL overexpression. STS (1 µM) was added for 24 hrs before cells were replated and colonies were stained with methylene blue typically 14 days after treatment.


G Quantification of colony formation of Bax / Bak DKO cells expressing Bax , BaxTBak , Bak or BakTBax with or without Bcl - xL overexpression after STS treatment ( Fig . 5F ) . Data represent averages ± SEM . n = 4 . p - values according to One Way ANOVA . BaxTBak expressing cells with or without Bcl - xL expression showed no significant difference ( n . s . ) . H Quantification of the colony formation of Bax / Bak DKO cells expressing Bax , BaxTBak , Bak or BakTBax in presence of Bcl - xL overexpression without apoptosis stimulation . Data represent averages ± SEM . n = 5 . p - values according to One Way ANOVA .

O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O 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-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 Quantification of colony formation of Bax/Bak DKO cells expressing Bax, BaxTBak, Bak or BakTBax with or without Bcl-xL overexpression after STS treatment (Fig. 5F). Data represent averages ± SEM. n = 4. p-values according to One Way ANOVA. BaxTBak expressing cells with or without Bcl-xL expression showed no significant difference (n.s.).H Quantification of the colony formation of Bax/Bak DKO cells expressing Bax, BaxTBak, Bak or BakTBax in presence of Bcl-xL overexpression without apoptosis stimulation. Data represent averages ± SEM. n = 5. p-values according to One Way ANOVA.


A FLIP of BaxTBakSS without ( ○ , broken line ) or with ( ● , straight line ) overexpressed Bcl - xL . Fluorescence of a neighboring cell is shown as control ( , dotted line ) . Data represent averages ± SEM from 20 ( - Bcl - xL ) and 16 ( + Bcl - xL ) ROI measurements .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O

A FLIP of BaxTBakSS without (○, broken line) or with (●, straight line) overexpressed Bcl-xL. Fluorescence of a neighboring cell is shown as control (, dotted line). Data represent averages ± SEM from 20 (-Bcl-xL) and 16 (+Bcl-xL) ROI measurements.


B Western blot analysis of PARP cleavage in HCT116 Bax / Bak DKO cells overexpressing Bax , Bax S184V , BaxTBak or BaxTBak V197 / 198S . The experiment was carried out in the absence of apoptotic stimuli . Cells transfected with pcDNA3 . 1 vector serve as control for PARP cleavage . Similar sample loading is controlled by actin . n = 4 .

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B Western blot analysis of PARP cleavage in HCT116 Bax/Bak DKO cells overexpressing Bax, Bax S184V, BaxTBak or BaxTBak V197/198S. The experiment was carried out in the absence of apoptotic stimuli. Cells transfected with pcDNA3.1 vector serve as control for PARP cleavage. Similar sample loading is controlled by actin. n = 4.


C Colony formation of Bax / Bak DKO cells transfected with pcDNA , BaxTBak or BaxTBakSS with Bcl - xL overexpression in the absence of apoptotic stimuli . n = 3 .

O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

C Colony formation of Bax/Bak DKO cells transfected with pcDNA, BaxTBak or BaxTBakSS with Bcl-xL overexpression in the absence of apoptotic stimuli. n = 3.


D Caspase 3 / 7 activity was measured in HCT116 Bax / Bak DKO cells overexpressing Bax , Bax S184V , BaxTBak or BaxTBak V197 / 198S . Caspase activity is displayed in relative fluorescence units ( RFU ) . The experiment was carried out in the absence of apoptotic stimuli and pcDNA3 . 1 - transfected cells served as control . Data represent averages ± SEM . n = 4 . p - values according to One Way ANOVA using the Holm - Sidak method are displayed .

O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Caspase 3/7 activity was measured in HCT116 Bax/Bak DKO cells overexpressing Bax, Bax S184V, BaxTBak or BaxTBak V197/198S. Caspase activity is displayed in relative fluorescence units (RFU). The experiment was carried out in the absence of apoptotic stimuli and pcDNA3.1-transfected cells served as control. Data represent averages ± SEM. n = 4. p-values according to One Way ANOVA using the Holm-Sidak method are displayed.


F STS - induced caspase 3 / 7 activity of HCT116 Bax / Bak DKO cells expressing Bax , Bax S184V , BakTBax or BakTBax S184V is displayed normalized to Bax activity . Data represent averages ± SEM . n ≥ 3 .

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F STS-induced caspase 3/7 activity of HCT116 Bax/Bak DKO cells expressing Bax, Bax S184V, BakTBax or BakTBax S184V is displayed normalized to Bax activity. Data represent averages ± SEM. n ≥ 3.


G Western blot analysis of PARP cleavage after STS treatment in HCT116 Bax / Bak DKO cells overexpressing Bax , Bax S184V , BakTBax or BakTBax S184V . Empty vector - transfected cells serve as control for PARP cleavage and actin is the loading control . n = 3 .

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G Western blot analysis of PARP cleavage after STS treatment in HCT116 Bax/Bak DKO cells overexpressing Bax, Bax S184V, BakTBax or BakTBax S184V. Empty vector-transfected cells serve as control for PARP cleavage and actin is the loading control. n = 3.


H Plot of Bax ( ● ) and Bak ( ○ ) retrotranslocation rates vs . hydrophobicity of the MA of the constructs . R2 = 0 . 95 .

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H Plot of Bax (●) and Bak (○) retrotranslocation rates vs. hydrophobicity of the MA of the constructs. R2 = 0.95.


A Carbonate extraction of the HM protein fraction of Bax / Bak DKO cells expressing wild - type Bcl - xL in the presence or the absence of Bak or Bax analyzed by Western blot . Carbonate extractable supernatant ( S ) and membrane - integral protein pellet ( P ) are displayed . Smac is released from the IMS during carbonate extraction and VDAC remains in the pellet . n = 4 . B Western blot analysis of Bcl - xLTBax carbonate extraction from the heavy membrane fraction from Bax / Bak DKO cells expressing the Bcl - xL variant with or without Bak or Bax . Smac and VDAC serve as controls for supernatant ( S ) and pellet ( P ) . n = 4 . C Carbonate extraction of the HM protein fraction of Bax / Bak DKO cells expressing Bcl - xL G138A with or without Bak or Bax . Smac and VDAC serve as controls for supernatant ( S ) and pellet ( P ) . n = 4 .

O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O

A Carbonate extraction of the HM protein fraction of Bax/Bak DKO cells expressing wild-type Bcl-xL in the presence or the absence of Bak or Bax analyzed by Western blot. Carbonate extractable supernatant (S) and membrane-integral protein pellet (P) are displayed. Smac is released from the IMS during carbonate extraction and VDAC remains in the pellet. n = 4.B Western blot analysis of Bcl-xLTBax carbonate extraction from the heavy membrane fraction from Bax/Bak DKO cells expressing the Bcl-xL variant with or without Bak or Bax. Smac and VDAC serve as controls for supernatant (S) and pellet (P). n = 4.C Carbonate extraction of the HM protein fraction of Bax/Bak DKO cells expressing Bcl-xL G138A with or without Bak or Bax. Smac and VDAC serve as controls for supernatant (S) and pellet (P). n = 4.


D HM protein fraction and carbonate extraction of Bax / Bak DKO cells expressing Bax , BaxTBak or Bax S184V . Smac and VDAC serve as controls for supernatant ( S ) and pellet ( P ) . n = 4 .

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D HM protein fraction and carbonate extraction of Bax/Bak DKO cells expressing Bax, BaxTBak or Bax S184V. Smac and VDAC serve as controls for supernatant (S) and pellet (P). n = 4.


E Smac release from the heavy membrane fraction ( HM ) into the cytosol ( C ) in HCT116 Bax / Bak DKO cells overexpressing Bax and BaxTBak without apoptotic stimuli analyzed by Western blot . Bax and BaxTBak were expressed differentially to produce similar levels of mitochondrial Bax . GAPDH and Tom20 serve as fractionation controls . n = 3 .

O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O

E Smac release from the heavy membrane fraction (HM) into the cytosol (C) in HCT116 Bax/Bak DKO cells overexpressing Bax and BaxTBak without apoptotic stimuli analyzed by Western blot. Bax and BaxTBak were expressed differentially to produce similar levels of mitochondrial Bax. GAPDH and Tom20 serve as fractionation controls. n = 3.


F PARP cleavage in HCT116 Bax / Bak DKO cells expressing similar levels of mitochondrial Bax or BaxTBak analyzed by Western blot without apoptotic stimuli . Actin is used as loading control . n = 3 .

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F PARP cleavage in HCT116 Bax/Bak DKO cells expressing similar levels of mitochondrial Bax or BaxTBak analyzed by Western blot without apoptotic stimuli. Actin is used as loading control. n = 3.


A Luciferase complementation imaging ( LCI ) assays showing that BIC1 interacts with BZR1 . nLUC - BZR1 was co - transformed with cLUC - CCA1 , cLUC - TOC1 , cLUC - ELF3 , cLUC - LUX , cLUC - BIC1 , cLUC , respectively , in leaf epidermal cells of Nicotiana benthamiana as indicated .

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A Luciferase complementation imaging (LCI) assays showing that BIC1 interacts with BZR1. nLUC-BZR1 was co-transformed with cLUC-CCA1, cLUC-TOC1, cLUC-ELF3, cLUC-LUX, cLUC-BIC1, cLUC, respectively, in leaf epidermal cells of Nicotiana benthamiana as indicated.


B BiFC assays showing the interaction of BIC1 and BZR1 mainly in nucleus . Leaf epidermal cells of Nicotiana benthamiana were co - transformed with nYFP - BZR1 and cYFP - BIC1 . BF , bright field . Scale bars represent 20 μm .

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B BiFC assays showing the interaction of BIC1 and BZR1 mainly in nucleus. Leaf epidermal cells of Nicotiana benthamiana were co-transformed with nYFP-BZR1 and cYFP-BIC1. BF, bright field. Scale bars represent 20 μm.


C In vitro pull - down assays showing BIC1 directly interacts with BZR1 . Purified GST - BIC1 proteins were incubated with MBP or MBP - BZR1 for the MBP pull - down assay . Arrowhead indicates specific bands .

O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O

C In vitro pull-down assays showing BIC1 directly interacts with BZR1. Purified GST-BIC1 proteins were incubated with MBP or MBP-BZR1 for the MBP pull-down assay. Arrowhead indicates specific bands.


D Co - immunoprecipitation ( Co - IP ) assays showing the interaction between BIC1 and BZR1 in vivo . Seedlings were grown for 6 days under long - day conditions . The immunoprecipitates were detected using anti - GFP and anti - MYC antibodies , respectively . * indicate specific bands . The cross - reacting lower band on IP blot is the heavy chain after IP .

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D Co-immunoprecipitation (Co-IP) assays showing the interaction between BIC1 and BZR1 in vivo. Seedlings were grown for 6 days under long-day conditions. The immunoprecipitates were detected using anti-GFP and anti-MYC antibodies, respectively. * indicate specific bands. The cross-reacting lower band on IP blot is the heavy chain after IP.


A , B Hypocotyl elongation phenotypes of the BIC1 overexpression and mutant lines in response to BR treatment . Col - 0 , 35S : BIC1 - YFP , 35S : BIC1 - Flag and bic1bic2 seedlings were grown for 6 days on 1 / 2 MS medium supplemented with 1 μM brassinazole ( BRZ ) plus different concentrations of epibrassinolide ( eBL ) . Images of the representative seedlings are shown in ( A ) , and the hypocotyl lengths of the indicated genotypes were measured and are shown in ( B ) . Data are means ± SD ; n > 20 . Scale bars , 2 mm .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, B Hypocotyl elongation phenotypes of the BIC1 overexpression and mutant lines in response to BR treatment. Col-0, 35S:BIC1-YFP, 35S:BIC1-Flag and bic1bic2 seedlings were grown for 6 days on 1/2 MS medium supplemented with 1 μM brassinazole (BRZ) plus different concentrations of epibrassinolide (eBL). Images of the representative seedlings are shown in (A), and the hypocotyl lengths of the indicated genotypes were measured and are shown in (B). Data are means ± SD; n>20. Scale bars, 2 mm.


C RT - qPCR analysis of BZR1 - activated gene expression in the bic1bic2 double mutants . The 6 - d - old Col - 0 and bic1bic2 seedlings grown on 1 / 2 MS medium supplemented with 1 μM BRZ were treated with 5 μM eBL ( + eBL ) or not ( - eBL ) for 1 hour . The PP2A gene served as internal control . Data are means ± SD ( n = 3 ) .

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C RT-qPCR analysis of BZR1-activated gene expression in the bic1bic2 double mutants. The 6-d-old Col-0 and bic1bic2 seedlings grown on 1/2 MS medium supplemented with 1 μM BRZ were treated with 5 μM eBL (+eBL) or not (-eBL) for 1 hour. The PP2A gene served as internal control. Data are means ± SD (n=3).


D Heat map of BIC1 - regulated genes . The Col - 0 and 35S : BIC1 - YFP seedlings were grown on 1 / 2 MS medium for 10 days . Three biologic replicates were performed . The colored bar beneath the map indicates fold change ( log2 value ) .

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D Heat map of BIC1-regulated genes. The Col-0 and 35S:BIC1-YFP seedlings were grown on 1/2 MS medium for 10 days. Three biologic replicates were performed. The colored bar beneath the map indicates fold change (log2 value).


E BIC1 facilitates the expression of cell elongation genes . RT - qPCR analysis of genes selected in transcriptomic data . The Col - 0 , 35S : BIC1 - YFP and 35S : BIC1 - Flag seedlings were grown for 5 days under long - day conditions . The PP2A gene served as internal control . Data are means ± SD ( n = 3 ) .

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E BIC1 facilitates the expression of cell elongation genes. RT-qPCR analysis of genes selected in transcriptomic data. The Col-0, 35S:BIC1-YFP and 35S:BIC1-Flag seedlings were grown for 5 days under long-day conditions. The PP2A gene served as internal control. Data are means ± SD (n=3).


A , B BIC1 and BZR1 synergistically promote hypocotyl elongation . The Col - 0 , 35S : BZR1 - MYC , 35S : BIC1 - YFP and 35S : BZR1 - MYC / 35S : BIC1 - YFP plants were grown for 5 days under long - day conditions . Images of the representative seedlings are shown in ( A ) , and the hypocotyl lengths of the indicated genotypes were measured and are shown in ( B ) . Data are means ± SD ( n > 20 ) . * * p < 0 . 01 , as determined by Student ' s t - test . Scale bar , 2 mm .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, B BIC1 and BZR1 synergistically promote hypocotyl elongation. The Col-0, 35S:BZR1-MYC, 35S:BIC1-YFP and 35S:BZR1-MYC/35S:BIC1-YFP plants were grown for 5 days under long-day conditions. Images of the representative seedlings are shown in (A), and the hypocotyl lengths of the indicated genotypes were measured and are shown in (B). Data are means ± SD (n>20). **p < 0.01, as determined by Student's t-test. Scale bar, 2 mm.


C - E BR signaling is enhanced in the 35S : BZR1 - MYC / 35S : BIC1 - YFP plants . Seedlings were grown for 6 days on medium supplemented with 1 μM brassinazole ( BRZ ) plus a gradient of concentrations of epibrassinolide ( eBL ) under long - day conditions . Images of the representative seedlings when grown with 500 nM eBL ( + eBL ) or not ( - eBL ) are shown in ( C ) , and the hypocotyl lengths of the indicated genotypes were measured and are shown in ( D ) and ( E ) . Data are means ± SD ( n > 20 ) . * * p < 0 . 01 , as determined by Student ' s t - test . Scale bar , 2 mm .

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C-E BR signaling is enhanced in the 35S:BZR1-MYC/35S:BIC1-YFP plants. Seedlings were grown for 6 days on medium supplemented with 1 μM brassinazole (BRZ) plus a gradient of concentrations of epibrassinolide (eBL) under long-day conditions. Images of the representative seedlings when grown with 500 nM eBL (+eBL) or not (-eBL) are shown in (C), and the hypocotyl lengths of the indicated genotypes were measured and are shown in (D) and (E). Data are means ± SD (n>20). **p < 0.01, as determined by Student's t-test. Scale bar, 2 mm.


F , G BZR1 - mediated hypocotyl elongation phenotype is dependent on the function of BIC1 and BIC2 . Seedlings were grown for 5 days under long - day conditions . Images of the representative seedlings are shown in ( F ) , and the hypocotyl lengths of the indicated genotypes were measured and are shown in ( G ) . Data are means ± SD ( n > 20 ) . * * p < 0 . 01 , n . s . indicates no significant difference , ( Student ' s t - test ) . Scale bar , 2 mm .

O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, G BZR1-mediated hypocotyl elongation phenotype is dependent on the function of BIC1 and BIC2. Seedlings were grown for 5 days under long-day conditions. Images of the representative seedlings are shown in (F), and the hypocotyl lengths of the indicated genotypes were measured and are shown in (G). Data are means ± SD (n>20). **p < 0.01, n.s. indicates no significant difference, (Student's t-test). Scale bar, 2 mm.


H BZR1 - mediated hypocotyl elongation phenotype is dependent on the function of BIC1 and BIC2 . The Col - 0 , 35S : BZR1 - MYC , bic1bic2 , 35S : BZR1 - MYC / bic1bic2 seedlings were grown on medium supplemented with 1 μM BRZ plus different concentrations of eBL for 6 days and then the hypocotyl lengths were measured . Data are means ± SD ( n > 20 ) .

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H BZR1-mediated hypocotyl elongation phenotype is dependent on the function of BIC1 and BIC2. The Col-0, 35S:BZR1-MYC, bic1bic2, 35S:BZR1-MYC/bic1bic2 seedlings were grown on medium supplemented with 1 μM BRZ plus different concentrations of eBL for 6 days and then the hypocotyl lengths were measured. Data are means ± SD (n>20).


A BR enhances the stability of BIC1 proteins . The 5 - d - old 35S : BIC1 - YFP transgenic plants were treated with 100 μM cycloheximide ( CHX ) or co - treated with 100 μM CHX and 1 μM epibrassinolide ( eBL ) . Samples were collected at indicated time points and BIC1 - YFP protein levels were analyzed by western blots using anti - GFP antibody . Actin was used to verify equal protein loadings . Intensity of the bands was measured using Adobe Photoshop CS3 Extended program . Three independent experiments were performed with similar results .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O 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 BR enhances the stability of BIC1 proteins. The 5-d-old 35S:BIC1-YFP transgenic plants were treated with 100 μM cycloheximide (CHX) or co-treated with 100 μM CHX and 1 μM epibrassinolide (eBL). Samples were collected at indicated time points and BIC1-YFP protein levels were analyzed by western blots using anti-GFP antibody. Actin was used to verify equal protein loadings. Intensity of the bands was measured using Adobe Photoshop CS3 Extended program. Three independent experiments were performed with similar results.


C Transactivation analysis of BIC1 using a yeast assays . The GAL4 DNA - binding domain ( BD ) alone was used as the negative control . SD - W , synthetic dextrose medium lacking Trp ; SD - WH , synthetic dextrose medium lacking both Trp and His .

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C Transactivation analysis of BIC1 using a yeast assays. The GAL4 DNA-binding domain (BD) alone was used as the negative control. SD-W, synthetic dextrose medium lacking Trp; SD-WH, synthetic dextrose medium lacking both Trp and His.


E Transient transcriptional activity analysis in Arabidopsis protoplasts illustrating the transcriptional activation activity of BIC1 . The relative luciferase activities were calculated by normalizing the LUC values against REN . Error bars represent SD ( n = 3 ) . * * p < 0 . 01 , as determined by a Student ' s t - test .

O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O 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 Transient transcriptional activity analysis in Arabidopsis protoplasts illustrating the transcriptional activation activity of BIC1. The relative luciferase activities were calculated by normalizing the LUC values against REN. Error bars represent SD (n = 3). **p < 0.01, as determined by a Student's t-test.


G Transient expression assays in Arabidopsis protoplasts showing that BIC1 and BZR1 synergistically activate PRE5 promoter . The ProPRE5 : LUC reporter was co - transformed with the indicated effector constructs . The LUC / REN ratio represents the ProPRE5 : LUC activity relative to the internal control ( REN driven by the 35S promoter ) . Error bars represent SD ( n = 3 ) .

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G Transient expression assays in Arabidopsis protoplasts showing that BIC1 and BZR1 synergistically activate PRE5 promoter. The ProPRE5:LUC reporter was co-transformed with the indicated effector constructs. The LUC/REN ratio represents the ProPRE5:LUC activity relative to the internal control (REN driven by the 35S promoter). Error bars represent SD (n = 3).


H , I BIC1 - mediated hypocotyl elongation was reduced by the addition of SRDX motif . Seedlings were grown for 5 days under long - day conditions . Images of the representative seedlings are shown in ( H ) , and the hypocotyl lengths of the indicated genotypes were measured and are shown in ( I ) . # 1 , # 2 , # 3 represent different 35S : BIC1 - SRDX - Flag transgenic lines . Data are means ± SD ( n > 20 ) . * * p < 0 . 01 , as determined by Student ' s t - test . Scale bar , 2 mm .

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H, I BIC1-mediated hypocotyl elongation was reduced by the addition of SRDX motif. Seedlings were grown for 5 days under long-day conditions. Images of the representative seedlings are shown in (H), and the hypocotyl lengths of the indicated genotypes were measured and are shown in (I). #1, #2, #3 represent different 35S:BIC1-SRDX-Flag transgenic lines. Data are means ± SD (n>20). **p < 0.01, as determined by Student's t-test. Scale bar, 2 mm.


LCI assays showing that BIC1 interacts with PIF4 . Leaf epidermal cells of Nicotiana benthamiana were co - transformed with nLUC - BIC1 or nLUC and cLUC - PIF4 or cLUC as indicated .

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LCI assays showing that BIC1 interacts with PIF4. Leaf epidermal cells of Nicotiana benthamiana were co-transformed with nLUC-BIC1 or nLUC and cLUC-PIF4 or cLUC as indicated.


B In vitro pull - down assays showing that BIC1 directly interacts with PIF4 . Purified GST - BIC1 proteins were incubated with MBP or MBP - PIF4 for the MBP pull - down assay . Arrowhead indicates specific bands .

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B In vitro pull-down assays showing that BIC1 directly interacts with PIF4. Purified GST-BIC1 proteins were incubated with MBP or MBP-PIF4 for the MBP pull-down assay. Arrowhead indicates specific bands.


C Co - IP assays showing the interaction between BIC1 and PIF4 in vivo . The 35S : BIC1 - YFP , 35S : PIF4 - MYC and 35S : PIF4 - MYC / 35S : BIC1 - YFP transgenic plants were grown for 6 days under long - day conditions . The immunoprecipitates were detected using anti - GFP and anti - MYC antibodies , respectively .

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C Co-IP assays showing the interaction between BIC1 and PIF4 in vivo. The 35S:BIC1-YFP, 35S:PIF4-MYC and 35S:PIF4-MYC/35S:BIC1-YFP transgenic plants were grown for 6 days under long-day conditions. The immunoprecipitates were detected using anti-GFP and anti-MYC antibodies, respectively.


D , E Phenotypic analyses showing that BIC1 and PIF4 synergistically promote hypocotyl elongation . The Col - 0 , 35S : BIC1 - YFP , 35S : PIF4 - MYC and 35S : PIF4 - MYC / 35S : BIC1 - YFP plants were grown for 5 days under long - day conditions . Images of the representative seedlings are shown in ( D ) , and the hypocotyl lengths of the indicated genotypes were measured and are shown in ( E ) . Data are means ± SD ( n > 20 ) . * * p < 0 . 01 , as determined by Student ' s t - test . Scale bar , 2 mm .

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D, E Phenotypic analyses showing that BIC1 and PIF4 synergistically promote hypocotyl elongation. The Col-0, 35S:BIC1-YFP, 35S:PIF4-MYC and 35S:PIF4-MYC/35S:BIC1-YFP plants were grown for 5 days under long-day conditions. Images of the representative seedlings are shown in (D), and the hypocotyl lengths of the indicated genotypes were measured and are shown in (E). Data are means ± SD (n>20). **p < 0.01, as determined by Student's t-test. Scale bar, 2 mm.


F RT - qPCR analysis showing that BIC1 and PIF4 synergistically activate PIF4 target genes . The seedlings growth conditions were same as ( D ) . The PP2A gene was used as an internal control . Data are means ± SD ( n = 3 ) .

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F RT-qPCR analysis showing that BIC1 and PIF4 synergistically activate PIF4 target genes. The seedlings growth conditions were same as (D). The PP2A gene was used as an internal control. Data are means ± SD (n=3).