( F ) Mouse bone marrow c - Kit + derived from Trp53 - / - mice were transduced with MLL - AF9 - GFP were transplanted into mice to generate p53 - deficient ( p53 - / - ) leukemia . GFP + MLL - AF9 leukemia were collected from bone marrows of vehicle - or FF - 10501 - 01 - treated wild - type p53 - / - leukemic mice . Expression of p53 Tubulin were assessed by western blotting 24 hours after treatment .

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

(F) Mouse bone marrow c-Kit+ cells derived from Trp53-/- mice were transduced with MLL-AF9-GFP and were transplanted into mice to generate p53-deficient (p53-/-) leukemia cells. GFP+ MLL-AF9 leukemia cells were collected from bone marrows of vehicle- or FF-10501-01-treated wild-type and p53-/- leukemic mice. Expression levels of p53 and Tubulin were assessed by western blotting 24 hours after the treatment.


( H ) Kaplan - Meier survival curves of p53 - / - MLL - AF9 leukemia mice treated with vehicle or FF - 10501 - 01or DS - 5272 ( n = 6 per group ) . Statistical significance was evaluated by log - rank ( left ) . Frequency of GFP + leukemia in peripheral blood at day 19 ( right , n = 6 per group ) . A two - unpaired t - was used for comparison . Data information : data are shown as mean ± .

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

(H) Kaplan-Meier survival curves of p53-/- MLL-AF9 leukemia mice treated with vehicle or FF-10501-01or DS-5272 (n = 6 per group). Statistical significance was evaluated by the log-rank test (left). Frequency of GFP+ leukemia cells in peripheral blood at day 19 (right, n = 6 per group). A two-tailed unpaired t-test was used for the comparison. Data information: All data are shown as mean ± SEM.


( B ) TLR - expressing BaκB were stimulated with corresponding TLR ligands ( 100 ng / mL Pam3CSK4 for TLR2 , 100 ng / mL LPS for TLR4 , 10 ng / mL Flagellin for TLR5 , 100 ng / ml R848 for TLR7 ) with titrating doses of MPA ( 1 - 10 μM ) . GFP expression was assessed 24 hours after stimulation .

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

(B) The TLR-expressing BaκB cells were stimulated with the corresponding TLR ligands (100 ng/mL Pam3CSK4 for TLR2, 100 ng/mL LPS for TLR4, 10 ng/mL Flagellin for TLR5, 100 ng/ml R848 for TLR7) with titrating doses of MPA (1 - 10 μM). GFP expression was assessed 24 hours after the stimulation.


( C ) 293T were transfected with FLAG - tagged TRAF6 HA - tagged K63 - ubiquitin . After 24 hours , these were treated with 3 or 10 μM MPA ( M - 3 μM or M - 10 μM ) with / without 100 μM guanosine ( G ) . IRAK1 / 4 inhibitor ( IRAK1 / 4 i ) was also used as a control . Whole - extracts were immunoprecipitated with anti - FLAG antibody , ubiquitinated TRAF6 was detected with anti - HA antibody .

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

(C) 293T cells were transfected with FLAG-tagged TRAF6 and HA-tagged K63-ubiquitin. After 24 hours, these cells were treated with 3 or 10 μM MPA (M-3 μM or M-10 μM) with/without 100 μM guanosine (G). IRAK1/4 inhibitor (IRAK1/4 i) was also used as a control. Whole-cell extracts were immunoprecipitated with anti-FLAG antibody, and ubiquitinated TRAF6 was detected with anti-HA antibody.


( D , Murine bone marrow - derived macrophages ( BMDMs ) were treated with vehicle , 10 μM MPA alone ( D ) N = 3 ( technical replicates ) per group . mRNA of TNF - α IL - 1β were measured by qPCR . Two - unpaired t - were used for comparison in ( D ) . Data information : data are shown as mean ± .

O O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE 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 B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD 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

(D, Murine bone marrow-derived macrophages (BMDMs) were treated with vehicle, 10 μM MPA alone (D) N=3 (technical replicates) per group. mRNA levels of TNF-α and IL-1β were measured by qPCR. Two-tailed unpaired t-tests were used for the comparison in (D). Data information: All data are shown as mean ± SEM.


E ) Murine bone marrow - derived macrophages ( BMDMs ) were treated with vehicle , 10 μM MPA alone or co - treated with vehicle , 10 μM MPA ( M ) , 100 ng / mL Pam3CSK4 100 μM Guanosine ( G ) for 4 hours ( E ) , as indicated . N = 3 ( technical replicates ) per group . mRNA of TNF - α IL - 1β were measured by qPCR . Ordinary one - way ANOVA was used for comparison in ( E ) . Data information : data are shown as mean ± .

O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE 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 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-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD 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

E) Murine bone marrow-derived macrophages (BMDMs) were treated with vehicle, 10 μM MPA alone or co-treated with vehicle, 10 μM MPA (M), 100 ng/mL Pam3CSK4 and 100 μM Guanosine (G) for 4 hours (E), as indicated. N=3 (technical replicates) per group. mRNA levels of TNF-α and IL-1β were measured by qPCR. Ordinary one-way ANOVA was used for the comparison in (E). Data information: All data are shown as mean ± SEM.


( F ) THP1 were pretreated with 10 μM MPA for 2 hours then treated with 1000 ng / mL Pam3CSK4 for 0 , 30 , 60 , ( 120 ) minutes . were lysed subjected to SDS - PAGE . of total IκBα , phosphate - p38 , total p38 , GAPDH were evaluated by western blotting .

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

(F) THP1 cells were pretreated with 10 μM MPA for 2 hours and then treated with 1000 ng/mL Pam3CSK4 for 0, 30, 60, and (120) minutes. Cells were lysed and subjected to SDS-PAGE. Levels of total IκBα, phosphate-p38, total p38, and GAPDH were evaluated by western blotting.


( G ) MLL - AF9 - expressing CB ( CB - MLL - AF9 # 1 ) were pretreated with 10 μM MPA for 2 hours then treated with 1000 ng / mL Pam3CSK4 for 0 , 30 , 60 were lysed subjected to SDS - PAGE . of total IκBα , phosphate - p38 , total p38 were evaluated by western blotting .

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

(G) MLL-AF9-expressing CB cells (CB-MLL-AF9#1) were pretreated with 10 μM MPA for 2 hours and then treated with 1000 ng/mL Pam3CSK4 for 0, 30, 60 Cells were lysed and subjected to SDS-PAGE. Levels of total IκBα, phosphate-p38, total p38 were evaluated by western blotting.


A . SDS - PAGE of purified T4SS3 - 10 + D4 . Left panel : Coomassie - stained SDS - PAGE gel of T4SS3 - 10 + D4 . Right panel : Sypro Ruby - stained SDS - PAGE gel of T4SS3 - 10m T4SS3 - 10 + D4 complexes . Molecular weights of MW markers are shown on left . Identification of bands is shown in middle . " * " indicates TrwK / VirB4 degradation products . " * * " indicates minor contaminants OmpA OmpC .

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

A. SDS-PAGE analysis of purified T4SS3-10+D4 complex. Left panel: Coomassie-stained SDS-PAGE gel of the T4SS3-10+D4 complex. Right panel: Sypro Ruby-stained SDS-PAGE gel of the T4SS3-10m and T4SS3-10+D4 complexes. Molecular weights of MW markers are shown on the left. Identification of bands is shown in the middle. "*" indicates TrwK/VirB4 degradation products. "**" indicates minor contaminants OmpA and OmpC.


B . Stoichiometry measurement . scan of an SDS - PAGE gel of T4SS3 - 10 + D4 where cysteine residues were reacted with AlexaFluor 633 C5 maleimide . Signals corresponding to coupled to AlexaFluor 633 were detected at a wavelength of 633 nm . Trw bands are labelled derived stoichiometry for TrwB / VirD4 is indicated .

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

B. Stoichiometry measurement. Fluorescence scan of an SDS-PAGE gel analysis of the T4SS3-10+D4 complex where cysteine residues were reacted with AlexaFluor 633 C5 maleimide. Signals corresponding to the proteins coupled to AlexaFluor 633 were detected at a wavelength of 633 nm. Fluorescent Trw protein bands are labelled and the derived stoichiometry for TrwB/VirD4 is indicated.


C . SDS - PAGE of complexes purified from expressing deletion mutants of pBADM11 _ trwN / virB1 - trwE / virB10Strep _ trwD / virB11 _ HistrwB / virD4 constructs termed pBADM11 _ ΔtrwX / ΔvirBY ( see main text ) . same molecular weights marker has been used on this gel as in gel presented in Figure 1A . * indicates degradation products of TrwK / VirB4 .

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

C. SDS-PAGE analysis of complexes purified from cell expressing deletion mutants of the pBADM11_trwN/virB1-trwE/virB10Strep_trwD/virB11_HistrwB/virD4 constructs termed pBADM11_ΔtrwX/ΔvirBY (see main text). The same molecular weights marker has been used on this gel as in the gel presented in Figure 1A. * indicates degradation products of TrwK/VirB4.


Figure 2 . Interaction between TrwB / VirD4 TrwE / VirB10 . Left panel : Coomassie - stained SDS - PAGE gel of T4SS3 - 10 + D4 ( lane 1 ) , T4SS3 - 10 + D4 : TrwEΔN42 ( lane 2 ) T4SS3 - 10 + D4 : TrwEΔN64 ( lane 3 ) . Middle panel : Western Blot using αStrep antibodies to detect TrwE / VirB10 within T4SS3 - 10 + D4 its variants . Right panel : Western Blot using αHis antibodies to detect TrwB / VirD4 within T4SS3 - 10 + D4 its variants .

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

Figure 2. Interaction between TrwB/VirD4 and TrwE/VirB10. Left panel: Coomassie-stained SDS-PAGE gel of the T4SS3-10+D4 complex (lane 1), T4SS3-10+D4:TrwEΔN42 complex (lane 2) and T4SS3-10+D4:TrwEΔN64 complex (lane 3). Middle panel: Western Blot analysis using αStrep antibodies to detect the TrwE/VirB10 protein within the T4SS3-10+D4 complex and its variants. Right panel: Western Blot analysis using αHis antibodies to detect the TrwB/VirD4 protein within the T4SS3-10+D4 complex and its variants.


A . NS - EM micrograph of T4SS3 - 10 + D4 . A few representative particles of T4SS3 - 10 + D4 are circled in yellow . Scale bar 50nm . B . Representative class averages of T4SS3 - 10 + D4 obtained after initial alignment classification .

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

A. NS-EM micrograph of the T4SS3-10+D4 complex. A few representative particles of the T4SS3-10+D4 complex are circled in yellow. Scale bar 50nm. B. Representative class averages of T4SS3-10+D4 complex obtained after initial alignment and classification.


C . upper row represents typical class averages of T4SS3 - 10 + D4 IMC ( i . e . with outer ( OMC ) masked out ) . second row shows projections of T4SS3 - 10 + D4 IMC in same directions determined for classes above . third row displays projections of T4SS3 - 10 IMC ( EMD - 2567 ) in same directions . bottom row shows differences between projections above ( plus or minus TrwB / VirD4 ) corresponding to positions of TrwB / VirD4 .

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

C. The upper row represents typical class averages of the T4SS3-10+D4 IMC complex (i.e. with the outer membrane core complex (OMC) masked out). The second row shows projections of the T4SS3-10+D4 IMC complex in the same directions determined for the classes above. The third row displays projections of the T4SS3-10 IMC complex (EMD-2567) in the same directions. The bottom row shows the differences between the projections above (plus or minus TrwB/VirD4) corresponding to positions of the TrwB/VirD4 protein.


D . FSC of electron density map of T4SS3 - 10 + D4 . 0 . 5 crosses FSC at a resolution of 28 Å .

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

D. FSC of the electron density map of the T4SS3-10+D4 complex. The 0.5 line crosses the FSC at a resolution of 28 Å.


A . Side views of NS - EM structure of T4SS3 - 10 ( left ) T4SS3 - 10 + D4 ( right ) complexes . arches , TrwK / VirB4 barrels , TrwB / VirD4 density are shown in green , yellow , blue , respectively . boundaries of 3 - tiers structure of TrwK / VirB4 barrel are indicated in middle of panels each tier is labelled as lower , middle , upper , respectively .

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

A. Side views of the NS-EM structure of the T4SS3-10 (left) and T4SS3-10+D4 (right) complexes. The arches, the TrwK/VirB4 barrels, and the TrwB/VirD4 density are shown in green, yellow, and blue, respectively. The boundaries of the 3-tiers structure of the TrwK/VirB4 barrel are indicated in the middle of the panels and each tier is labelled as lower, middle, and upper, respectively.


B . Side view of T4SS3 - 10 + D4 structure ( upper panel ) derived schematic representation ( lower panel ) . Overall dimensions are indicated C . Same as in B except that side - view shown is rotated 90 degrees along a vertical axis compared to view shown in panel B . Overall dimension is indicated . D . Bottom view of NS - EM structure of T4SS3 - 10 ( upper panel ) corresponding schematic representation ( lower panel ) . Angle between axes connecting TrwK / VirB4 hexamer pair TrwB / VirD4 dimer pair is shown .

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

B. Side view of the T4SS3-10+D4 structure (upper panel) and derived schematic representation (lower panel). Overall dimensions are indicated C. Same as in B except that the side-view shown is rotated 90 degrees along a vertical axis compared to the view shown in panel B. Overall dimension is indicated. D. Bottom view of the NS-EM structure of the T4SS3-10 (upper panel) and the corresponding schematic representation (lower panel). Angle between the axes connecting the TrwK/VirB4 hexamer pair and the TrwB/VirD4 dimer pair is shown.


B . Class averages of antibody - bound T4SS3 - 10 + D4FLAG . Class averages of aligned NS - EM of T4SS3 - 10 + D4FLAG with 3 - 4 particles per class . Red circles indicate position of antibody .

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

B. Class averages of antibody-bound T4SS3-10+D4FLAG. Class averages of the aligned NS-EM images of the T4SS3-10+D4FLAG with 3-4 particles per class. Red circles indicate the position of the antibody.


A , B growth curve ( left ) summary of end - point masses ( right ) of 6 - 8 week - old wildtype ( WT ) Peli1 - KO ( KO ) mice inoculated s . c . with B16F10 ( A ; WT , n = 7 ; KO , n = 6 ) or E . G7 ( B ; WT , n = 5 ; KO , n = 5 ) .

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

A,B Tumor growth curve (left) and summary of end-point tumor masses (right) of 6-8 week-old wildtype (WT) and Peli1-KO (KO) mice inoculated s.c. with B16F10 (A; WT, n=7; KO, n=6) or E.G7 (B; WT, n=5; KO, n=5) tumor cells.


C , D Flow cytometry of CD4 + CD8 + T in CD45 . 2 + TILs dLN from B16F10 - bearing wildtype Peli1 - KO mice , presented as a representative plot ( C ) summary graph on multiple mice ( D ; WT , n = 6 ; KO , n = 6 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O O B-CELL_LINE O O O B-GENEPROD O O B-ORGANISM 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 O O O O

C,D Flow cytometry analysis of CD4+ and CD8+ T cells in CD45.2+ TILs and dLN cells from B16F10 tumor-bearing wildtype and Peli1-KO mice, presented as a representative plot (C) and summary graph based on multiple mice (D; WT, n=6; KO, n=6).


E , F Flow cytometry of IFNγ - producing CD4 + CD8 + T in TILs , dLN of B16F10 - bearing ( day 19 ) wildtype Peli1 - KO mice , presented as a representative plot ( E ) summary graph ( F ) . ( WT , n = 6 ; KO , n = 6 )

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

E,F Flow cytometry analysis of IFNγ-producing CD4+ and CD8+ T cells in TILs, and dLN cells of B16F10 tumor-bearing (day 19) wildtype and Peli1-KO mice, presented as a representative plot (E) and summary graph (F). (WT, n=6; KO, n=6)


G , H Flow cytometry of IFNγ + CD8 + T in TILs of E . G7 - bearing ( day 24 ) wildtype Peli1 - KO mice , presented as a representative plot ( G ) summary graph ( H ) . ( WT , n = 3 ; KO , n = 3 )

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

G,H Flow cytometry analysis of IFNγ+CD8+ T cells in TILs of E.G7 tumor-bearing (day 24) wildtype and Peli1-KO mice, presented as a representative plot (G) and summary graph (H). (WT, n=3; KO, n=3)


A , B growth curve ( left ) summary graph of end - point masses ( right ) of wildtype ( WT ) Peli1 - TKO ( TKO ) mice inoculated s . c . with B16 - OVA ( A ; WT , n = 5 ; KO , n = 5 ) or E . G7 ( B ; WT , n = 6 ; KO , n = 4 ) .

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

A,B Tumor growth curve (left) and summary graph of end-point tumor masses (right) of wildtype (WT) and Peli1-TKO (TKO) mice inoculated s.c. with B16-OVA (A; WT, n=5; KO, n=5) or E.G7 (B; WT, n=6; KO, n=4) tumor cells.


C Flow cytometry of IFNγ - producing CD4 + CD8 + T in TILs dLN from B16OVA - bearing wildtype Peli1 - TKO mice , presented as a representative plot ( upper ) summary graph ( lower ) . ( WT , n = 5 ; KO , n = 5 )

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C Flow cytometry analysis of IFNγ-producing CD4+ and CD8+ T cells in TILs and dLN cells from B16OVA tumor-bearing wildtype and Peli1-TKO mice, presented as a representative plot (upper) and summary graph (lower). (WT, n=5; KO, n=5)


D Flow cytometry of GZMB + CD8 + T in TILs from B16 - OVA - bearing wildtype Peli1 - TKO mice , presented as a representative plot ( upper ) summary graph ( lower ) . ( WT , n = 5 ; KO , n = 5 )

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D Flow cytometry analysis of GZMB+CD8+ T cells in TILs from B16-OVA tumor-bearing wildtype and Peli1-TKO mice, presented as a representative plot (upper) and summary graph (lower). (WT, n=5; KO, n=5)


E growth curve ( left ) summary graph of end - point masses ( right ) of B16 - OVA - bearing wildtype indicated Peli1 conditional KO mice . ( Treg KO model : WT , n = 5 ; Treg KO , n = 6 ; MKO model : WT , n = 3 ; MKO , n = 5 ; BKO model : WT , n = 9 ; BKO , n = 9 )

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E Tumor growth curve (left) and summary graph of end-point tumor masses (right) of B16-OVA tumor-bearing wildtype and the indicated Peli1 conditional KO mice. (Treg KO model: WT, n=5; Treg KO, n=6; MKO model: WT, n=3; MKO, n=5; BKO model: WT, n=9; BKO, n=9)


A , B Seahorse of basal ( glucose injection ) maximal ( oligomycin injection ) ECAR or basal ( no treatment ) maximal ( FCCP injection ) OCR in WT Peli1 - KO OT - I CD8 T that were either untreated ( naïve ) or activated with plate - bound anti - CD3 ( 1 μg / ml ) plus anti - CD28 ( 1 μg / ml ) for 16 h . Data are presented as a representative plot ( A ) or summary graph on 6 wildtype 6 KO mice ( B ) .

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A,B Seahorse analysis of basal (glucose injection) and maximal (oligomycin injection) ECAR or basal (no treatment) and maximal (FCCP injection) OCR in WT and Peli1-KO OT-I CD8 T cells that were either untreated (naïve) or activated with plate-bound anti-CD3 (1 μg/ml) plus anti-CD28 (1 μg/ml) for 16 h. Data are presented as a representative plot (A) or summary graph based on 6 wildtype and 6 KO mice (B).


qRT - PCR of indicated genes using RNAs isolated from wildtype or Peli1 - KO OT - I CD8 T stimulated with anti - CD3 anti - CD28 for 6 h .

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qRT-PCR analysis of the indicated genes using RNAs isolated from wildtype or Peli1-KO OT-I CD8 T cells stimulated with anti-CD3 and anti-CD28 for 6 h.


D qRT - PCR of indicated genes using RNAs isolated from wildtype or Peli1 - KO OT - I CD8 T stimulated with anti - CD3 anti - CD28 for 6 h .

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D qRT-PCR analysis of the indicated genes using RNAs isolated from wildtype or Peli1-KO OT-I CD8 T cells stimulated with anti-CD3 and anti-CD28 for 6 h.


E Immunoblot of HK2 Glut1 in wildtype or Peli1 - KO OT - I CD8 T that were stimulated with plate - bound anti - CD3 anti - CD28 for 24 h .

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E Immunoblot analysis of HK2 and Glut1 in wildtype or Peli1-KO OT-I CD8 T cells that were stimulated with plate-bound anti-CD3 and anti-CD28 for 24 h.


A Immunoblot of indicated phosphorylated ( p - ) total in whole - lysates of freshly isolated CD8 T from wildtype ( WT ) or Peli1 KO ( KO ) mice ( 6 old ) . Data are presented as a representative blot ( left ) summary graphs of densitometric quantifications of indicated ( presented as phosphorylated / total ) on three independent experiments .

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A Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of freshly isolated CD8 T cells from wildtype (WT) or Peli1 KO (KO) mice (6 weeks old). Data are presented as a representative blot (left) and summary graphs of densitometric quantifications of the indicated proteins (presented as phosphorylated/total protein ratio) based on three independent experiments.


B , C Immunoblot of indicated phosphorylated ( p - ) total in whole - lysates of wildtype or Peli1 - KO OT - I CD8 T that were first rested on ice for 15 min then stimulated in low - serum ( 1 % FBS ) medium with anti - CD3 anti - CD28 for indicated time periods .

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B,C Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of wildtype or Peli1-KO OT-I CD8 T cells that were first rested on ice for 15 min and then stimulated in low-serum (1% FBS) medium with anti-CD3 and anti-CD28 for the indicated time periods.


D , E Seahorse of basal maximal ECAR ( D ) or OCR ( E ) using wildtype or Peli1 - KO OT - I CD8 T that were activated for 16 h with plate - bound anti - CD3 ( 1 μg / ml ) plus anti - CD28 ( 1 μg / ml ) in presence of solvent control ( DMSO ) , rapamycin ( Rapa , 10 nM ) , or Torin 1 ( 100 nM ) . Data are presented as a representative plot ( left ) summary graphs ( right ) .

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

D,E Seahorse analysis of basal and maximal ECAR (D) or OCR (E) using wildtype or Peli1-KO OT-I CD8 T cells that were activated for 16 h with plate-bound anti-CD3 (1 μg/ml) plus anti-CD28 (1 μg/ml) in the presence of solvent control (DMSO), rapamycin (Rapa, 10 nM), or Torin 1 (100 nM). Data are presented as a representative plot (left) and summary graphs (right).


F , G Immunoblot of indicated phosphorylated ( p - ) total in whole - lysates of wildtype or Peli1 - KO OT - I CD8 T that were either not treated ( - ) or stimulated ( + ) for 1 h ( F ) or 24 h ( G ) with anti - CD3 ( 1 μg / ml ) anti - CD28 ( 1 μg / ml ) in presence ( + ) or absence ( - ) of rapamycin ( 10 nM ) Torin 1 ( 100 nM ) .

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F,G Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of wildtype or Peli1-KO OT-I CD8 T cells that were either not treated (-) or stimulated (+) for 1 h (F) or 24 h (G) with anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) in the presence (+) or absence (-) of rapamycin (10 nM) and Torin 1 (100 nM).


H qRT - PCR of Ifng gene expression in naïve wildtype or Peli1 - KO OT - I CD8 T that were either not treated ( - ) or stimulated for 6 h ( + ) with anti - CD3 plus anti - CD28 in presence ( + ) or absence ( - ) or rapamycin .

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H qRT-PCR analysis of Ifng gene expression in naïve wildtype or Peli1-KO OT-I CD8 T cells that were either not treated (-) or stimulated for 6 h (+) with anti-CD3 plus anti-CD28 in the presence (+) or absence (-) or rapamycin.


I - K Schematic of experimental design ( I ) , growth curve ( J ) , summary of day 15 ( K ) of B6 . SJL mice inoculated with B16 - OVA melanoma ( 2 x 105 ) adoptively transferred on day 7 with in vitro activated rapamycin - or DMSO - treated wildtype or Peli1 - KO OT - I CD8 T ( 4 x 105 ) .

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I-K Schematic of experimental design (I), tumor growth curve (J), and summary of day 15 tumor weight (K) of B6.SJL mice inoculated with B16-OVA melanoma cells (2 x 105) and adoptively transferred on day 7 with in vitro activated and rapamycin- or DMSO-treated wildtype or Peli1-KO OT-I CD8 T cells (4 x 105).


Immunoblot of indicated phosphorylated ( p - ) total in whole - lysates of wildtype ( WT ) or Peli1 - KO ( KO ) OT - I CD8 T that were activated for 16 h with plate - coated anti - CD3 ( 1 μg / ml ) plus anti - CD28 , starved for 6 h in serum - free RPMI medium , then treated for indicated time periods with FBS ( 10 % ) ( A )

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Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of wildtype (WT) or Peli1-KO (KO) OT-I CD8 T cells that were activated for 16 h with plate-coated anti-CD3 (1 μg/ml) plus anti-CD28, starved for 6 h in serum-free RPMI medium, and then treated for indicated time periods with FBS (10%) (A)


Immunoblot of indicated phosphorylated ( p - ) total in whole - lysates of wildtype ( WT ) or Peli1 - KO ( KO ) OT - I CD8 T that were activated for 16 h with plate - coated anti - CD3 ( 1 μg / ml ) plus anti - CD28 , starved for 6 h in serum - free RPMI medium , then treated for indicated time periods with insulin ( B ) .

B-EXP_ASSAY O O B-EXP_ASSAY 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 B-CELL_TYPE 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 B-GENEPROD O O O O O O B-TISSUE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O

Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of wildtype (WT) or Peli1-KO (KO) OT-I CD8 T cells that were activated for 16 h with plate-coated anti-CD3 (1 μg/ml) plus anti-CD28, starved for 6 h in serum-free RPMI medium, and then treated for indicated time periods with insulin (B).


Immunoblot of indicated phosphorylated ( p - ) total in whole - lysates of wildtype ( WT ) or Peli1 - KO ( KO ) MEFs that were starved for 16 h in serum - free DMEM medium then treated for indicated time periods with FBS ( C )

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

Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of wildtype (WT) or Peli1-KO (KO) MEFs that were starved for 16 h in serum-free DMEM medium and then treated for the indicated time periods with FBS (C)


Immunoblot of indicated phosphorylated ( p - ) total in whole - lysates of wildtype ( WT ) or Peli1 - KO ( KO ) MEFs that were starved for 16 h in serum - free DMEM medium then treated for indicated time periods with insulin ( D )

B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-CELL_TYPE O O O O O O O B-TISSUE O O O O O O O O O O O B-SMALL_MOLECULE O O O

Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of wildtype (WT) or Peli1-KO (KO) MEFs that were starved for 16 h in serum-free DMEM medium and then treated for the indicated time periods with insulin (D)


Immunoblot of indicated phosphorylated ( p - ) total in whole - lysates of wildtype ( WT ) or Peli1 - KO ( KO ) MEFs that were starved for 16 h in serum - free DMEM medium then treated for indicated time periods with EGF ( E ) .

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

Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of wildtype (WT) or Peli1-KO (KO) MEFs that were starved for 16 h in serum-free DMEM medium and then treated for the indicated time periods with EGF (E).


A Immunoblot of indicated in whole - lysates of wildtype ( WT ) Peli1 - KO ( KO ) CD8 T that were either not treated ( NT ) or activated for 16 h with plate - bound anti - CD3 anti - CD28 . Data are presented as a representative blot ( left panel ) a summary graph of quantified TSC1 ( middle panel ) TSC2 ( right panel ) bands relative to level of α - Tubulin .

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

A Immunoblot analysis of the indicated proteins in whole-cell lysates of wildtype (WT) and Peli1-KO (KO) CD8 T cells that were either not treated (NT) or activated for 16 h with plate-bound anti-CD3 and anti-CD28. Data are presented as a representative blot (left panel) and a summary graph of quantified TSC1 (middle panel) and TSC2 (right panel) protein bands relative to the level of α-Tubulin.


Immunoblot of indicated in whole - lysates of wildtype Peli1 - KO CD8 T that were activated for 1 h with anti - CD3 anti - CD28 then treated with cycloheximide ( CHX ) for indicated time periods . Data are presented as a representative blot ( B )

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

Immunoblot analysis of the indicated proteins in whole-cell lysates of wildtype and Peli1-KO CD8 T cells that were activated for 1 h with anti-CD3 and anti-CD28 and then treated with cycloheximide (CHX) for the indicated time periods. Data are presented as a representative blot (B)


a summary graph of quantified TSC1 TSC2 bands presented as of level in untreated ( 0 min ) ( C ) .

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

a summary graph of quantified TSC1 and TSC2 protein bands presented as percentage of the level in untreated (0 min) cells (C).


D Immunoblot of TSC1 TSC2 in wildtype or Peli1 - KO CD8 T stimulated with anti - CD3 plus anti - CD28 for 1 h then incubated with ( + ) or without ( - ) cycloheximide ( CHX ) / or MG132 for 2 h . Data are presented as a representative blot ( left panel ) a summary graph of quantified TSC1 ( middle panel ) TSC2 ( right panel ) bands relative to level of α - Tubulin .

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D Immunoblot analysis of TSC1 and TSC2 in wildtype or Peli1-KO CD8 T cells stimulated with anti-CD3 plus anti-CD28 for 1 h and then incubated with (+) or without (-) cycloheximide (CHX) and/or MG132 for 2 h. Data are presented as a representative blot (left panel) and a summary graph of quantified TSC1 (middle panel) and TSC2 (right panel) protein bands relative to the level of α-Tubulin.


Ubiquitination of immunoprecipitated TSC2 ( E ) from activated ( anti - CD3 plus anti - CD28 for 2 h ) wildtype or Peli1 - KO CD8 T by immunobloting using anti - ubiquitin Abs detecting total ( Ub ) , K48 ( Ub - K48 ) , or K63 ( Ub - K63 ) polyubiquitin chains .

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Ubiquitination analysis of immunoprecipitated TSC2 (E) from activated (anti-CD3 plus anti-CD28 for 2 h) wildtype or Peli1-KO CD8 T cells by immunobloting using anti-ubiquitin Abs detecting total (Ub), K48 (Ub-K48), or K63 (Ub-K63) polyubiquitin chains.


Ubiquitination of immunoprecipitated TSC1 ( F ) from activated ( anti - CD3 plus anti - CD28 for 2 h ) wildtype or Peli1 - KO CD8 T by immunobloting using anti - ubiquitin Abs detecting total ( Ub ) , K48 ( Ub - K48 ) , or K63 ( Ub - K63 ) polyubiquitin chains .

Ubiquitination analysis of immunoprecipitated TSC1 (F) from activated (anti-CD3 plus anti-CD28 for 2 h) wildtype or Peli1-KO CD8 T cells by immunobloting using anti-ubiquitin Abs detecting total (Ub), K48 (Ub-K48), or K63 (Ub-K63) polyubiquitin chains.


of ubiquitin conjugation to TSC1 ( G , H ) in 293T transiently transfected ( for 48 h ) with indicated expression vectors .

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Analysis of ubiquitin conjugation to TSC1 (G, H) in 293T cells transiently transfected (for 48 h) with the indicated expression vectors.


of ubiquitin conjugation to TSC1 TSC2 ( I , J ) in 293T transiently transfected ( for 48 h ) with indicated expression vectors .

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Analysis of ubiquitin conjugation to TSC1 TSC2 (I, J) in 293T cells transiently transfected (for 48 h) with the indicated expression vectors.


K Co - immunoprecipitaton of TSC1 - TSC2 interaction in 293T transiently transfected ( for 48 h ) with indicated expression vectors .

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K Co-immunoprecipitaton analysis of TSC1-TSC2 interaction in 293T cells transiently transfected (for 48 h) with the indicated expression vectors.


L Co - immunoprecipitation of endogenous TSC1 - TSC2 interaction in wildtype Peli1 - KO CD8 T that were either not treated ( NT ) or stimulated for 2 h with anti - CD3 plus anti - CD28 . right panel is a summary graph of TSC2 / TSC1 on of blot from anti - TSC1 IP ( right four lanes of top two panels ) .

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L Co-immunoprecipitation analysis of endogenous TSC1-TSC2 interaction in wildtype and Peli1-KO CD8 T cells that were either not treated (NT) or stimulated for 2 h with anti-CD3 plus anti-CD28. The right panel is a summary graph of TSC2/TSC1 ratio based on quantification of the blot from anti-TSC1 IP (the right four lanes of the top two panels).


A of ubiquitin conjugation to wildtype ( WT ) TSC1 indicated TSC1 mutants in 293T transiently transfected Flag - tagged TSC1 its mutants along with ( + ) or without ( - ) indicated expression vectors .

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A Analysis of ubiquitin conjugation to wildtype (WT) TSC1 and the indicated TSC1 mutants in 293T cells transiently transfected Flag-tagged TSC1 and its mutants along with (+) or without (-) the indicated expression vectors.


B Immunoblot of indicated phosphorylated ( p - ) total in lysates of TSC1 - KO MEFs transfected with ( + ) or without ( - ) TSC1 wildtype or K30A expression vectors . were serum starved then left either untreated ( NT ) or stimulated with 10 % FBS for 30 mins . Data are presented as a representative blot ( left panel ) a summary graph of quantified TSC2 ( right panel ) bands relative to level of β - Actin .

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B Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in lysates of TSC1-KO MEFs transfected with (+) or without (-) TSC1 wildtype or K30A expression vectors. The cells were serum starved and then left either untreated (NT) or stimulated with 10% FBS for 30 mins. Data are presented as a representative blot (left panel) and a summary graph of quantified TSC2 (right panel) protein bands relative to the level of β-Actin.


C of TSC2 ubiquitination in 293 transfected ( for 48 h ) with ( + ) or without ( - ) indicated expression vectors .

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C Analysis of TSC2 ubiquitination in 293 cells transfected (for 48 h) with (+) or without (-) the indicated expression vectors.


D Co - immunoprecipitation of interaction between endogenous TSC2 exogenous TSC1 wildtype K30A in TSC1 - KO MEFs transfected ( for 48 h ) with ( + ) or without ( - ) indicated expression vectors . KO MEF were transfected with indicated expression plasmids . right panel is a summary graph of TSC2 / TSC1 on of last four lanes of anti - Flag IP blot in left panel .

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D Co-immunoprecipitation analysis of the interaction between endogenous TSC2 and exogenous TSC1 wildtype and K30A in TSC1-KO MEFs transfected (for 48 h) with (+) or without (-) the indicated expression vectors. KO MEF cells were transfected with indicated expression plasmids. The right panel is a summary graph of TSC2/TSC1 ratio based on quantification of the last four lanes of the anti-Flag IP blot in left panel.


E of ubiquitin conjugation to endogenous TSC2 in TSC1 - KO MEFs transfected ( for 48 h ) with ( + ) or without ( - ) TSC1 WT or K30A treated with MG132 for 2 h before lysis .

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E Analysis of ubiquitin conjugation to endogenous TSC2 in TSC1-KO MEFs transfected (for 48 h) with (+) or without (-) TSC1 WT or K30A and treated with MG132 for 2 h before lysis.


F Immunoblot of endogenous TSC2 in TSC1 - KO MEFs transfected ( for 48 h ) with Myc - TSC1 wildtype or K30A subsequently treated with cycloheximide ( 10 μg / ml ) for indicated time periods before lysis . right panel is a summary graph of quantified TSC2 WT K30A bands presented as percentages of level in untreated ( 0 min ) lane .

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F Immunoblot analysis of endogenous TSC2 in TSC1-KO MEFs transfected (for 48 h) with Myc-TSC1 wildtype or K30A and subsequently treated with cycloheximide (10 μg/ml) for the indicated time periods before lysis. The right panel is a summary graph of quantified TSC2 WT and K30A protein bands presented as percentages of the level in the untreated (0 min) lane.


C - E . 2a 2b ESCs maintained in 2i medium were cultured in absence ( Ctrl ) or presence of OHT subsequently used for WB ( C )

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C-E. 2a and 2b ESCs maintained in 2i medium were cultured in the absence (Ctrl) or presence of OHT and subsequently used for WB (C)


C - E . 2a 2b ESCs maintained in 2i medium were cultured in absence ( Ctrl ) or presence of OHT subsequently used for growth curves RT - qPCR ( D , E ) .

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C-E. 2a and 2b ESCs maintained in 2i medium were cultured in the absence (Ctrl) or presence of OHT and subsequently used for growth curves and RT-qPCR (D,E).


F - G . 2a 2b ESCs were adapted to grow in serum - containing ESC medium , exposed to OHT as indicated subsequently used for growth curves RT - qPCR analyses .

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F-G. 2a and 2b ESCs were adapted to grow in serum-containing ESC medium, exposed to OHT as indicated and subsequently used for growth curves and RT-qPCR analyses.


H - I . Jmjd2a ( + / - ) ; Jmjd2a ( + / - ) ( H ) or Jmjd2b ( + / - ) ; Jmjd2b ( + / - ) ( I ) mice were inter - crossed number of pups alive at time of weaning reported . P values were calculated using a Chi - square .

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H-I. Jmjd2a(+/-); Jmjd2a(+/-) (H) or Jmjd2b(+/-); Jmjd2b(+/-) (I) mice were inter-crossed and the number of pups alive at time of weaning reported. P values were calculated using a Chi-square test.


A - D . WB growth curves of 2ac ( A ) , 2abc ( B ) , 2ab ( C ) 2bc ( D ) ESCs cultured in 2i medium exposed to OHT as indicated .

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A-D. WB and growth curves of 2ac (A), 2abc (B), 2ab (C) and 2bc (D) ESCs cultured in 2i medium and exposed to OHT as indicated.


E . Flow cytometry profiles of 2ac ESCs pulsed with BrdU in 2i medium . upper table shows percentages of living ( subG1 fraction is omitted from ) , which can be classified as being in S ( BrdU + ) , G1 ( 2N content , BrdU - ) or G2 / M phase ( 4N content , BrdU - ) . percentages of dead ( subG1 , events with a < 2N content ) is shown in red .

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E. Flow cytometry profiles of 2ac ESCs pulsed with BrdU in 2i medium. The upper table shows the percentages of living cells (the subG1 fraction is omitted from the analysis), which can be classified as being in S (BrdU+), G1 (2N DNA content, BrdU-) or G2/M phase (4N DNA content, BrdU-). The percentages of dead cells (subG1, events with a <2N DNA content) is shown in red.


F . RT - qPCR analyses for ESCs cultured in 2i medium .

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F. RT-qPCR analyses for ESCs cultured in 2i medium.


G - J . 2ac 2abc ESCs were adapted to grow in serum - containing ESC medium , exposed to OHT as indicated subsequently used for growth curves ( G - H ) RT - qPCR analyses ( I - J ) . RT - qPCR data are also presented in Appendix Fig S2C - D with a zoom - in on lower region of y - axis .

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G-J. 2ac and 2abc ESCs were adapted to grow in serum-containing ESC medium, exposed to OHT as indicated and subsequently used for growth curves (G-H) and RT-qPCR analyses (I-J). The RT-qPCR data are also presented in Appendix Fig S2C-D with a zoom-in on the lower region of the y-axis.


A - B . 2ac ESCs expressing GFP were cultured in absence or presence of OHT subsequently used for morula injections . ( A ) Average volume of GFP - positive in chimeric at 24 72 hrs after morula injection . Number of used for : Ctrl 24h n = 18 ; + OHT 24h n = 15 ; Ctrl 72h n = 5 ; + OHT 72h n = 5 . Data are presented as mean ± SD . ( B ) Representative of chimeric . bars indicate a of 32 µm . Relative intensities of GFP signal are indicated for each image .

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A-B. 2ac ESCs expressing GFP were cultured in the absence or presence of OHT and subsequently used for morula injections. (A) Average volume of the GFP-positive area in chimeric embryos at 24 and 72 hrs after morula injection. Number of embryos used for quantification: Ctrl 24h n=18; +OHT 24h n=15; Ctrl 72h n=5; +OHT 72h n=5. Data are presented as mean ± SD. (B) Representative images of chimeric embryos. The white bars indicate a length of 32 µm. Relative intensities of the GFP signal are indicated for each image.


C - D . Jmjd2a ( + / - ) ; Jmjd2c ( + / - ) mice were inter - crossed . number of ( C ) pups alive at weaning or ( D ) recovered at E6 . 5 is presented . P values were calculated using exact binomial with most conservative estimate of expected ( - / - ; - / - ) ( 6 . 25 % , assuming no linkage between Jmjd2a Jmjd2cloci even though they are located on same chromosome ) .

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C-D. Jmjd2a(+/-); Jmjd2c(+/-) mice were inter-crossed. The number of (C) pups alive at weaning or (D) embryos recovered at E6.5 is presented. P values were calculated using exact binomial test with the most conservative estimate of expected (-/-;-/-) embryos (6.25 %, assuming no linkage between the Jmjd2a and Jmjd2cloci even though they are located on the same chromosome).


A . Examples of ChIP - seq data obtained for Jmjd2a , Jmjd2c ( Pedersen et al , 2014 ) , H3K4me3 .

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A. Examples of ChIP-seq data obtained for Jmjd2a, Jmjd2c (Pedersen et al, 2014), and H3K4me3.


C . Unsupervised k - means clustering of ChIP - seq tags over TSSs ( + / - 5kb ) .

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C. Unsupervised k-means clustering of ChIP-seq tags over all TSSs (+/- 5kb).


D - E . Bar graphs showing number of Jmjd2a peaks displaying at least 1 bp overlap with ( D ) an H3K4me3 peak . Jmjd2a peaks were classified as TSS - associated ( ' TSS ' ) if overlapping a region of + / - 1kb of a TSS .

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D-E. Bar graphs showing the number of Jmjd2a peaks displaying at least 1 bp overlap with (D) an H3K4me3 peak. Jmjd2a peaks were classified as TSS-associated ('TSS') if overlapping a region of +/- 1kb of a TSS.


D - E . Bar graphs showing number of Jmjd2a peaks displaying at least 1 bp overlap with ( E ) a Jmjd2c peak . Jmjd2a peaks were classified as TSS - associated ( ' TSS ' ) if overlapping a region of + / - 1kb of a TSS .

D-E. Bar graphs showing the number of Jmjd2a peaks displaying at least 1 bp overlap with (E) a Jmjd2c peak. Jmjd2a peaks were classified as TSS-associated ('TSS') if overlapping a region of +/- 1kb of a TSS.


G . ChIP - qPCR validations . Graphs show mean ± SD for 3 technical replicates are representative of results obtained in at least 2 independent experiments .

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G. ChIP-qPCR validations. Graphs show mean ± SD for 3 technical replicates and are representative of results obtained in at least 2 independent experiments.


A . Density plots showing average ChIP - seq signals with 95 % confidence intervals of mean indicated in grey . y - axes show mean Tags per Million ( TPM ) . Data were obtained using 2ac ESCs # 4 . TSSs were classified as ' Bound ' if containing binding sites for both Jmjd2a Jmjd2c within + / - 1kb ( see Fig 4F ) , or ' Not bound ' if neither binds within same region .

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A. Density plots showing average ChIP-seq signals with 95% confidence intervals of the mean indicated in grey. y-axes show mean Tags per Million (TPM). Data were obtained using the 2ac ESCs line #4. TSSs were classified as 'Bound' if containing binding sites for both Jmjd2a and Jmjd2c within +/- 1kb (see Fig 4F), or 'Not bound' if neither protein binds within the same region.


B . Heat map showing H3K9me3 ChIP - seq data for Jmjd2a / c bound TSSs ( + / - 5kb ) sorted according to read number in 2abc # 13 + OHT .

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B. Heat map showing H3K9me3 ChIP-seq data for Jmjd2a/c bound TSSs (+/- 5kb) sorted according to read number in 2abc #13+OHT.


C . ChIP - qPCR validations for selected Jmjd2a / c targets ( see Fig 4G ) . Graphs show mean ± SD for technical triplicates are representative of results obtained in at least 2 independent experiments .

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C. ChIP-qPCR validations for selected Jmjd2a/c targets (see Fig 4G). Graphs show mean ± SD for technical triplicates and are representative of results obtained in at least 2 independent experiments.


A - B . Independent RT - qPCR validations of genes identified as being ( A ) down - regulated or ( B ) up - regulated by expression arrays . Graphs show mean ± SD for technical triplicates are representative of results obtained with at least 2 different ESCs lines of each genotype in independent experiments .

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A-B. Independent RT-qPCR validations of genes identified as being (A) down-regulated or (B) up-regulated by expression arrays. Graphs show mean ± SD for technical triplicates and are representative of results obtained with at least 2 different ESCs lines of each genotype in independent experiments.


C - D . Density plots showing average ChIP - seq signals for ( C ) H3K9me3 or ( D ) H3K36me3 with 95 % confidence intervals of mean indicated in grey . Plots are shown for genes , which are down - or up - regulated according to microarray analyses ( FDR < 0 . 05 , absolute fold change log2 ( | FC | ) > 0 . 5 ) for both 2ac 2abc KO ESCs ( overlaps presented in Appendix Fig S6A ) , or do not show strong expression changes ( log2 ( | FC | ) < 0 . 2 ) . Genes are further classified as " Bound " if containing binding sites for both Jmjd2a Jmjd2c within + / - 1kb of a TSS .

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C-D. Density plots showing average ChIP-seq signals for (C) H3K9me3 or (D) H3K36me3 with 95% confidence intervals of the mean indicated in grey. Plots are shown for genes, which are down- or up-regulated according to the microarray analyses (FDR<0.05, absolute fold change log2(|FC|)>0.5) for both 2ac and 2abc KO ESCs (overlaps presented in Appendix Fig S6A), or do not show strong expression changes (log2(|FC|) < 0.2). Genes are further classified as "Bound" if containing binding sites for both Jmjd2a and Jmjd2c within +/- 1kb of a TSS.


E . Heat map presenting array data in form of log2 ( fold change ) values comparing OHT with control treated ESCs . Genes are shown for which TSS regions ( + / - 1kb ) show most substantial increase in H3K9me3 in OHT treated according to ChIP - seq analyses ( defined in Fig EV4 ) .

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E. Heat map presenting array data in the form of log2(fold change) values comparing OHT with control treated ESCs. Genes are shown for which TSS regions (+/- 1kb) show the most substantial increase in H3K9me3 levels in OHT treated cells according to the ChIP-seq analyses (defined in Fig EV4).


A - C . 2ac clones established after transfection with an empty vector ( empty ) or plasmids expressing wild type Jmjd2c ( 2c wt ) or a catalytic mutant ( 2c mut ) were exposed to OHT as indicated subsequently used for ( A ) WB .

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A-C. 2ac clones established after transfection with an empty vector (empty) or plasmids expressing wild type Jmjd2c (2c wt) or a catalytic mutant (2c mut) were exposed to OHT as indicated and subsequently used for (A) WB.


A - C . 2ac clones established after transfection with an empty vector ( empty ) or plasmids expressing wild type Jmjd2c ( 2c wt ) or a catalytic mutant ( 2c mut ) were exposed to OHT as indicated subsequently used for ( B ) RT - qPCR

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A-C. 2ac clones established after transfection with an empty vector (empty) or plasmids expressing wild type Jmjd2c (2c wt) or a catalytic mutant (2c mut) were exposed to OHT as indicated and subsequently used for (B) RT-qPCR analyses


A - C . 2ac clones established after transfection with an empty vector ( empty ) or plasmids expressing wild type Jmjd2c ( 2c wt ) or a catalytic mutant ( 2c mut ) were exposed to OHT as indicated subsequently used for ( C ) serial plating for generation of growth curves .

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A-C. 2ac clones established after transfection with an empty vector (empty) or plasmids expressing wild type Jmjd2c (2c wt) or a catalytic mutant (2c mut) were exposed to OHT as indicated and subsequently used for (C) serial plating for the generation of growth curves.


a , Polychromasia ( Wright - Giemsa stain , indicated by arrows ) increased reticulocytes ( brilliant cresyl blue stain ) revealed by blood smears from 6 - week - old wild - type ( WT ) or Nix - / - mice ( scale bar , 20 µm ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-CELL_TYPE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O

a, Polychromasia (Wright-Giemsa stain, indicated by arrows) and increased reticulocytes (brilliant cresyl blue stain) revealed by staining blood smears from 6-week-old wild-type (WT) or Nix-/- mice (scale bar, 20 µm).


b , c , Ter119 ( b ) or Mitotracker deep red ( c ) versus CD71 of RBCs from 3 - or 6 - week - old mice . were also calculated ( mean ± s . e . m . ; n = 3 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 .

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b, c, Ter119 (b) or Mitotracker deep red (c) versus CD71 staining of RBCs from 3- or 6-week-old mice. Cell counts were also calculated (mean ± s.e.m.; n = 3). *P < 0.05; **P < 0.01; ***P < 0.001.


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d , Analyses of RBCs from 3 - or 6 - week - oldmice by

d, Analyses of RBCs from 3- or 6-week-oldmice by immunocytochemistry


Analyses of RBCs from 3 - or 6 - week - oldmice by transmission electron

Analyses of RBCs from 3- or 6-week-oldmice by transmission electron microscopy


a , of NHS - biotin - labelled RBCs or transferred CMFDA - labelled RBCs ( n = 3 ) .

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a, Quantification of NHS-biotin-labelled RBCs or transferred CMFDA-labelled RBCs (n = 3).


b , Western blot analyses of caspases in RBCs after in vitro culture . Asterisks denote processed caspases . FL , full .

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b, Western blot analyses of caspases in RBCs after in vitro culture. Asterisks denote processed caspases. FL, full length.


c , relative luminescence units ( RLU ) of caspase activities in cultured RBCs suppression of annexin V in Nix - / - RBCs after 12 - h culture in presence of qVD - oph or solvent control ( DMSO ) ( n = 3 ) .

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c, The relative luminescence units (RLU) of caspase activities in cultured RBCs and the suppression of annexin V staining in Nix-/- RBCs after 12-h culture in the presence of qVD-oph or solvent control (DMSO) (n = 3).


d , Mitotracker versus annexin V of RBCs after in vitro culture .

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d, Mitotracker versus annexin V staining of RBCs after in vitro culture.


e , Mean ( MFI ) of ROS in RBCs after in vitro culture , caspase activities in Nix - / - RBCs after 24 h culture with solvent control , ROS scavengers or qVD - oph ( n = 3 ) . BHT , butylated hydroxytoluene ; CAT , catalase ; , tempol .

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e, Mean fluorescent intensity (MFI) of ROS staining in RBCs after in vitro culture, and caspase activities in Nix-/- RBCs after 24 h culture with solvent control, ROS scavengers or qVD-oph (n = 3). BHT, butylated hydroxytoluene; CAT, catalase; TEM, tempol.


f , Haematoxylin eosin ( HE ) of spleen sections of 9 - week - old wild - type Nix - / - mice . Arrows denote iron deposits within macrophagecytoplasm . Iron deposits were also stained with Prussian blue , followed by counterstain with - fast red . Scale bar , 20 µm .

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f, Haematoxylin and eosin (HE) staining of spleen sections of 9-week-old wild-type and Nix-/- mice. Arrows denote iron deposits within macrophagecytoplasm. Iron deposits were also stained with Prussian blue, followed by counterstain with nuclear-fast red. Scale bar, 20 µm.


g , Real - time RT - PCR of erythropoietin ( Epo ) ( n = 12 ) . For relevant panels , statistical significance of data ( mean ± s . e . m . ) is : * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 .

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g, Real-time RT-PCR of erythropoietin (Epo) (n = 12). For all relevant panels, statistical significance of the data (mean ± s.e.m.) is: *P < 0.05; **P < 0.01; ***P < 0.001.


a , Ter119 + CD71 + reticulocytes sorted at day 3 or 6 after PHZ treatment were cultured for in vitro maturation for 0 ( top row ) , 2 ( middle row ) or 4 ( bottom row ) days , followed by Mitotracker deep red Ter119 .

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a, Ter119+CD71+ reticulocytes sorted at day 3 or 6 after PHZ treatment were cultured for in vitro maturation for 0 (top row), 2 (middle row) or 4 (bottom row) days, followed by Mitotracker deep red and Ter119 staining.


b , CD71 + Ter119 + reticulocytes sorted at day 3 after PHZ treatment were cultured stained with new methylene blue . Scale bar , 20 µm .

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b, CD71+Ter119+ reticulocytes sorted at day 3 after PHZ treatment were cultured and stained with new methylene blue. Scale bar, 20 µm.


c , CD71 + Ter119 + reticulocytes sorted at day 3 after PHZ treatment were stained for LC3 COX IV analysed by deconvolution microscopy . Scale bar , 5 µm . Pearson coefficiency for LC3 COX IV co - localization at day 0 ( mean ± s . e . m . ) is : wild type , 0 . 81 ± 0 . 001 ; Nix - / - , 0 . 44 ± 0 . 036 ( n = 35 , P = 0 . 0006 ) .

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

c, CD71+Ter119+ reticulocytes sorted at day 3 after PHZ treatment were stained for LC3 and COX IV and analysed by deconvolution microscopy. Scale bar, 5 µm. The Pearson coefficiency for LC3 and COX IV co-localization at day 0 (mean ± s.e.m.) is: wild type, 0.81 ± 0.001; Nix-/-, 0.44 ± 0.036 (n = 35, P = 0.0006).


d , CD71 + Ter119 + reticulocytes sorted at day 3 after PHZ treatment were analysed by electron microscopy . Scale bar , 0 . 5 µm .

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

d, CD71+Ter119+ reticulocytes sorted at day 3 after PHZ treatment were analysed by electron microscopy. Scale bar, 0.5 µm.


a , Ter119 + CD71 + reticulocytes were sorted from peripheral blood of wild - type Nix - / - mice at day 6 after PHZ treatment . were cultured for in vitro maturation in presence of 10 µM FCCP or 1 µM ABT - 737 , followed by with Mitotracker deep red phycoerythrin - conjugated anti - Ter119 .

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

a, Ter119+CD71+ reticulocytes were sorted from the peripheral blood of wild-type and Nix-/- mice at day 6 after PHZ treatment. The cells were cultured for in vitro maturation in the presence of 10 µM FCCP or 1 µM ABT-737, followed by staining with Mitotracker deep red and phycoerythrin-conjugated anti-Ter119.


b , c , Reticulocytes were cultured with FCCP ( b ) or ABT - 737 ( c ) for 24 h analysed by transmission electron microscopy . Scale bar , 0 . 5 µm .

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b, c, Reticulocytes were cultured with FCCP (b) or ABT-737 (c) for 24 h and analysed by transmission electron microscopy. Scale bar, 0.5 µm.


RNAi injections schedule in starved conditions . Planarians are at 14dS when amputation is performed ( indicated by grey cross at day 14 ) . Live show that cct3A ( RNAi ) planarians form a minimal blastema compared to controls at time points shown . At bottom are number of planarians with phenotype shown . remaining planarians are dead by time point of regeneration shown . dR indicates days of regeneration . RNAi injections schedule in feeding conditions . One extra feeding in respect to ( B ) is introduced one day prior to injections . Planarians are at 8dS when amputation is performed . live show that most of cct3A ( RNAi ) planarians regenerate like controls . At bottom are number of planarians with phenotype shown . At 8 - 10dR , remaining planarians are either dead ( 4 / 124 ) or have a tiny blastema ( 23 / 124 ) either died or regenerated later . At 51 - 68dR , remaining planarians are either dead ( 10 / 93 ) or have a tiny blastema died later ( 5 / 93 ) .

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

RNAi injections schedule in starved conditions. Planarians are at 14dS when the amputation is performed (indicated by the grey cross at day 14). Live images show that cct3A(RNAi) planarians form a minimal blastema compared to controls at the time points shown. At the bottom are the number of planarians with the phenotype shown. The remaining planarians are dead by the time point of regeneration shown. dR indicates days of regeneration. RNAi injections schedule in feeding conditions. One extra feeding in respect to (B) is introduced one day prior to injections. Planarians are at 8dS when the amputation is performed. The live images show that most of the cct3A(RNAi) planarians regenerate like controls. At the bottom are the number of planarians with the phenotype shown. At 8-10dR, the remaining planarians are either dead (4/124) or have a tiny blastema (23/124) and either died or regenerated later. At 51-68dR, the remaining planarians are either dead (10/93) or have a tiny blastema and died later (5/93).


graph shows mitotic numbers of trunks during different time points of regeneration in starved condition for cct3A RNAi controls . Error bars are s . d . from mean asterisks indicate P < 0 . 001 ( three asterisks ) , P < 0 . 01 ( two asterisks ) n . s . indicates not significant using two - Student ' s with equal variance . n ≥ 9 planarians per time point . Also shown are maximum projections of representative trunks labelled with anti - H3P at different time points of regeneration in starved condition . On bottom , number of planarians with phenotype shown from total is displayed . Note that at 50dR planarians show few ( a4 ) or no mitoses ( a5 ) . dashed delimits of planarian .

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 O O 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 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 O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O B-ORGANISM O

The graph shows the mitotic numbers of trunks during different time points of regeneration in the starved condition for cct3A RNAi and controls. Error bars are s.d. from the mean and asterisks indicate P < 0.001 (three asterisks), P < 0.01 (two asterisks) and n.s. indicates not significant using two-tailed Student's test with equal variance. n ≥ 9 planarians per time point. Also shown are maximum projections of representative trunks labelled with anti-H3P at different time points of regeneration in the starved condition. On the bottom, the number of planarians with the phenotype shown from the total is displayed. Note that at 50dR planarians show few (a4) or no mitoses (a5). The dashed line delimits the body of the planarian.


Maximum projections of representative trunks after FISH for smedwi - 1 at different time points of regeneration under starved feeding conditions . On bottom , number of planarians with phenotype shown from total is displayed . At 15dR expression are similar . At 30dR some planarians have almost no expression ( c5 ) at 50dR almost planarians show no expression ( c10 c11 ) in starved conditions . Under feeding condition expression at 30dS are similar . On bottom , number of planarians with phenotype shown from total is displayed . dashed delimits of planarian .

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

Maximum projections of representative trunks after FISH for smedwi-1 at different time points of regeneration under starved and feeding conditions. On the bottom, the number of planarians with the phenotype shown from the total is displayed. At 15dR the expression levels are similar. At 30dR some planarians have almost no expression (c5) and at 50dR almost all planarians show no expression (c10 and c11) in starved conditions. Under the feeding condition the expression levels at 30dS are similar. On the bottom, the number of planarians with the phenotype shown from the total is displayed. The dashed line delimits the body of the planarian.


cartoon represents a regenerating trunk squares display region analyzed at anterior " A " posterior " P " blastemas . are TUNEL maximum projections of representative blastemas from 72 hours 4 days regenerating trunks in starved conditions . dashed delimits of planarian . graph shows number of TUNEL + per mm2 . differences in are not significant ( n . s . ) at 72hR ( two - Student ' s with equal sample variance ) significant ( * * * P < 0 . 001 using two - Student ' s with equal variance ) at 4dR . n ≥ 5 planarians per condition .

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The cartoon represents a regenerating trunk and the squares display the region analyzed at the anterior "A" and posterior "P" blastemas. The images are TUNEL maximum projections of representative blastemas from 72 hours and 4 days regenerating trunks in starved conditions. The dashed line delimits the body of the planarian. The graph shows the number of TUNEL+ cells per mm2. The differences in cell death are not significant (n.s.) at 72hR (two-tailed Student's test with equal sample variance) and significant (***P < 0.001 using two-tailed Student's test with equal variance) at 4dR. n ≥ 5 planarians per condition.


Volcano plot displaying DEGs in starved cct3A ( RNAi ) planarians compared to controls at 72hR ( q - value < 0 . 05 ) . Y - axis indicates negative log10 of false discovery rate ( FDR ) ( q - value ) . X - axis indicates beta values ( b ) , a biased estimator of fold change . For better visualization , 13 dots with - log10 ( q - value ) > 20 are not displayed . This includes cct3A with q = 0 . Notice that when down - regulating cct3A in starved planarians , xbp1 , atf6 , bip - 1 a repertoire of ER chaperones are down - regulated , whereas dnajb9 , a repressor of UPR , other ccts are up - regulated .

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

Volcano plot displaying DEGs in starved cct3A(RNAi) planarians compared to controls at 72hR (q-value < 0.05). Y-axis indicates the negative log10 of the false discovery rate (FDR) (q- value). X-axis indicates the beta values (b), a biased estimator of the fold change. For better visualization, 13 dots with -log10 (q-value) > 20 are not displayed. This includes cct3A with q = 0. Notice that when down-regulating cct3A in starved planarians, xbp1, atf6, bip-1 and a repertoire of ER chaperones are down-regulated, whereas dnajb9, a repressor of the UPR, and other ccts are up-regulated.


Live show that xbp1 / atf6 ( RNAi ) planarians in starved conditions form a minimal blastema when compared to controls while at feeding condition most of them can regenerate as controls . remaining planarians in starved condition are dead by time point of regeneration shown . In feeding condition , remaining planarians at 8 - 10dR are either dead ( 13 / 145 ) or have a tiny blastema died or regenerated later ( 40 / 145 ) while at 21 - 27dR are dead .

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Live images show that xbp1/atf6(RNAi) planarians in starved conditions form a minimal blastema when compared to controls while at the feeding condition most of them can regenerate as controls. The remaining planarians in the starved condition are dead by the time point of regeneration shown. In the feeding condition, the remaining planarians at 8-10dR are either dead (13/145) or have a tiny blastema and died or regenerated later (40/145) while at 21-27dR are dead.


Maximum projections of representative regenerating trunks after FISH for smedwi - 1 in starved conditions . dashed delimits of planarian .

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Maximum projections of representative regenerating trunks after FISH for smedwi-1 in starved conditions. The dashed line delimits the body of the planarian.


TUNEL maximum projections of representative blastemas from regenerating trunks in starved conditions . dashed delimits of planarian . graph shows number of TUNEL + per mm2 at same time points in anterior ( A ) posterior ( P ) blastemas . n ≥ 6 planarians per time point .

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TUNEL maximum projections of representative blastemas from regenerating trunks in starved conditions. The dashed line delimits the body of the planarian. The graph shows the number of TUNEL+ cells per mm2 at the same time points in anterior (A) and posterior (P) blastemas. n ≥ 6 planarians per time point.


of of in different mitotic phases of defective mitotic figures in 72hR anterior blastemas of xbp1 / atf6 RNAi controls in starved conditions after double immunostaining with anti - tyrosine - tubulin anti - H3P ( * * * * P < 0 . 0001 , * * * P < 0 . 001 , * P < 0 . 05 , n . s . indicates not significant using two - sided Chi - square ) ; n ≥ 5 planarians . Representative are shown . Arrows indicate abnormal organization or number of spindle poles . j8 displays asymmetrical distribution of chromosome content .

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

Quantification of the percentage of stem cells in different mitotic phases and the percentage of defective mitotic figures in 72hR anterior blastemas of xbp1/atf6 RNAi and controls in starved conditions after double immunostaining with anti-tyrosine-tubulin and anti-H3P (****P < 0.0001, ***P < 0.001, *P < 0.05, n.s. indicates not significant using two-sided Chi-square test); n ≥ 5 planarians. Representative images are shown. Arrows indicate abnormal organization or number of spindle poles. j8 displays asymmetrical distribution of chromosome content.


Kaplan - Meier curve demonstrates increased survival of cct3A RNAi when treated with DTT under starved conditions . Percentages indicate number of survivals at indicated time points . Three asterisks indicate p < 0 . 001 with log - rank ( Mantel - Cox ) . are representative surviving for different conditions at displayed time point of regeneration . At bottom are number of planarians with phenotype shown . of planarians are dead at displayed time point .

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The Kaplan-Meier curve demonstrates increased survival of cct3A RNAi animals when treated with DTT under starved conditions. Percentages indicate the number of survivals at the indicated time points. Three asterisks indicate p < 0.001 with log-rank (Mantel-Cox) test. Images are representative surviving animals for the different conditions at the displayed time point of regeneration. At the bottom are the number of planarians with the phenotype shown. The rest of planarians are dead at the displayed time point.


Volcano plots displaying shRNA - Cct3 - shRNA - luciferase - infected LSK cultured either in normal glucose ( upper graph ) or low glucose conditions ( lower graph ) . Dots represent NanoString analyzed transcripts . Red dots are UPR ER stress related genes . UPR ER stress genes significantly down - regulated only under low glucose conditions in shCct3 compared to controls are indicated near lower graph . For better visualization two dots with q ≤ 0 . 001 have been removed from volcano plot ( see them in Dataset EV4 ) . Y - axis indicates false discovery rate ( FDR ) ( q - value ) , X - axis indicates log2 fold changes . FDR - method of p - value adjustment was conducted to calculate q - values by nSolver software using Benjamini - Hochberg methods . Significance is established by q - value < 0 . 05 indicated by dotted horizontal . n = 3 mice .

O O O O O B-GENEPROD O O O B-GENEPROD O O B-CELL_TYPE O O O O B-SMALL_MOLECULE 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 B-SUBCELLULAR O O O O O B-SUBCELLULAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM O

Volcano plots displaying shRNA-Cct3- and shRNA-luciferase-infected LSK cells cultured either in normal glucose (upper graph) or low glucose conditions (lower graph). Dots represent all the NanoString analyzed transcripts. Red dots are UPR and ER stress related genes. The UPR and ER stress genes significantly down-regulated only under low glucose conditions in shCct3 cells compared to controls are indicated near the lower graph. For better visualization two dots with q ≤ 0.001 have been removed from the volcano plot (see them in Dataset EV4). Y-axis indicates the false discovery rate (FDR) (q- value), and the X-axis indicates the log2 fold changes. The FDR-based method of p-value adjustment was conducted to calculate the q-values by nSolver software using Benjamini-Hochberg methods. Significance is established by q-value < 0.05 and indicated by the dotted horizontal line. n= 3 mice.


Kaplan - Meier curve demonstrates increased survival of starved cct3A ( RNAi ) when treated with palmitic acid / palmitoylcarnitine ( PA / PC ) . Percentages indicate number of survivals at indicated time points . Three asterisks indicate P < 0 . 001 with log - rank ( Mantel - Cox ) .

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The Kaplan-Meier curve demonstrates increased survival of starved cct3A(RNAi) animals when treated with palmitic acid/palmitoylcarnitine (PA/PC). Percentages indicate the number of survivals at the indicated time points. Three asterisks indicate P < 0.001 with log-rank (Mantel-Cox) test.


( A ) Western blot of SMC3 - LAP expressing HeLa treated with siRNA as indicated synchronized in G2 - phase . Efficiency of interference SMC3 acetylation were analyzed from total extracts after fractionation ( asterisks indicate unspecific signals that were depleted from antibody dilutions used in later experiments , presumably by repeated usage of antibody samples ) . Note that ESCO1 were increased on after depletion of ESCO2 , raising possibility that a decrease in bound ESCO2 might be compensated for by ESCO1 recruitment to .

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(A) Western blot of SMC3-LAP expressing HeLa cells treated with siRNA as indicated and synchronized in G2-phase. Efficiency of RNA interference and SMC3 acetylation levels were analyzed from total extracts and after chromatin fractionation (asterisks indicate unspecific signals that were depleted from antibody dilutions used in later experiments, presumably by repeated usage of the antibody samples). Note that ESCO1 levels were increased on chromatin after depletion of ESCO2, raising the possibility that a decrease in chromatin bound ESCO2 might be compensated for by ESCO1 recruitment to chromatin.


( B ) Still of an inverse recovery after photobleaching ( iFRAP ) experiment . intensities are false - colored as indicated . bar , 5 μm . ( C ) Graph depicting normalized after photobleaching from two biological replicates to quantify dissociation kinetics . Error bars denote standard error of mean ( s . e . m . ) , n = 10 per condition . ( D ) of relative abundance of SMC3 - LAP in stable binding mode .

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

(B) Still images of an inverse fluorescence recovery after photobleaching (iFRAP) experiment. Fluorescence intensities are false-colored as indicated. Size bar, 5 μm.(C) Graph depicting the normalized intensity after photobleaching from two biological replicates to quantify chromatin dissociation kinetics. Error bars denote standard error of the mean (s.e.m.), n = 10 cells per condition.(D) Quantification of the relative abundance of SMC3-LAP in the stable chromatin binding mode.


( A ) of iFRAP experiments using HeLa expressing SMC3 - LAP in wild - type form ( wt ) or with lysines 105 - 106 mutated to glutamines ( QQ ) or arginines ( RR ) synchronized in G1 - phase . Error bars denote s . e . m . , n > 25 per condition . ( B ) of residence time of SMC3 - LAP alleles in G1 - phase .

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(A) Quantification of iFRAP experiments using HeLa cells expressing SMC3-LAP in the wild-type form (wt) or with lysines 105-106 mutated to glutamines (QQ) or arginines (RR) and synchronized in G1-phase. Error bars denote s.e.m., n > 25 cells per condition.(B) Quantification of chromatin residence time of SMC3-LAP alleles in G1-phase.


( C ) of iFRAP experiments using synchronized in G2 - phase . Error bars denote s . e . m . , n > 26 per condition . ( D ) of stably bound SMC3 - LAP alleles in G2 - phase .

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(C) Quantification of iFRAP experiments using cells synchronized in G2-phase. Error bars denote s.e.m., n > 26 cells per condition.(D) Quantification of stably bound SMC3-LAP alleles in G2-phase.


( E ) Western blot of mouse fibroblasts expressing SMC3 - LAP in presence or absence of endogenous Smc3 after gene deletion . Extracts were prepared after subculturing two times . without SMC3 - LAP do not proliferate after deletion of Smc3 .

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(E) Western blot of mouse fibroblasts expressing SMC3-LAP in the presence or absence of endogenous Smc3 after gene deletion. Extracts were prepared after subculturing two times. Cells without SMC3-LAP do not proliferate after deletion of Smc3.


( F ) Western blot showing HeLa extracts after treatment with siRNA as indicated synchronized in G2 - phase . Note that ESCO1 immunoblot signals were also reduced in sororin depleted . We currently do not know if this is an effect of sororin depletion , an off - target effect or an artefact of unequal sample loading .

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

(F) Western blot showing HeLa cell extracts after treatment with siRNA as indicated and synchronized in G2-phase. Note that ESCO1 immunoblot signals were also reduced in sororin depleted cells. We currently do not know if this is an effect of sororin depletion, an off-target effect or an artefact of unequal sample loading.


( G ) iFRAP of stably bound SMC3 - LAP after siRNA treatment using synchronized in G2 - phase . Error bars denote s . e . m . , n > 11 per condition .

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(G) iFRAP quantification of stably chromatin bound SMC3-LAP after siRNA treatment using cells synchronized in G2-phase. Error bars denote s.e.m., n > 11 cells per condition.


( A ) FACS profile of transfected with nondegradable KEN - box mutant sororin - FLAG ( sorKBM - FLAG ) after synchronization in G1 - phase propidium iodide .

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(A) FACS profile of cells transfected with nondegradable KEN-box mutant sororin-FLAG (sorKBM-FLAG) after synchronization in G1-phase and propidium iodide staining.


( B ) Immunofluorescence microscopy experiment to determine transfection efficiency .

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(B) Immunofluorescence microscopy experiment to determine transfection efficiency.


( C ) Western blot of fractionated lysates to detect sororinKBM - FLAG .

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(C) Western blot of fractionated lysates to detect sororinKBM-FLAG.


( D ) of SMC3 - LAP residence time on after transfecting with sorKBM - FLAG in G1 - phase . Error bars denote s . e . m . ; n > 15 per condition .

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(D) Quantification of SMC3-LAP residence time on chromatin after transfecting cells with sorKBM-FLAG in G1-phase. Error bars denote s.e.m.; n > 15 cells per condition.


( E - F ) of cycle distribution transfection efficiency using synchronized in G2 - phase . ( G ) of stably - bound SMC3 - LAP from expressing nondegradable sororin in G2 - phase . were treated with siRNA as indicated .

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(E-F) Analysis of cell cycle distribution and transfection efficiency using cells synchronized in G2-phase.(G) Quantification of stably chromatin-bound SMC3-LAP from cells expressing nondegradable sororin in G2-phase. Cells were treated with siRNA as indicated.


( H ) Western blot showing fractionated nuclease digested from synchronized in G1 G2 - phase after immunoprecipitation with FLAG antibodies .

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(H) Western blot showing fractionated and nuclease digested chromatin from cells synchronized in G1 and G2-phase after immunoprecipitation with FLAG antibodies.


( A ) Examples of sororin ( dotted arrows ) SMC3ChIP - seq BrdU DIP - seq data from early S - phase as compared to G2 - phase using Integrated Genome Browser ( IGB ; Nicol et al , 2009 ) . ( B ) Alignment of sororin , BrdU BrdU - negative control sequencing bins in early S - phase to humanchromosome 18 ( GRCh37 / hg19 ) . Bars correspond to called peaks , curves to read enrichments after smoothing ( k = 25 kbp ) . ( C ) of co - occupancy of BrdU incorporation sororin localization in early S - phase sororin in G2 - phase depicted as p - value distributions calculated with IntervalStats ( Chikina Troyanskaya , 2012 ) compared to randomized controls . Sororin peaks were identified as overlapping peaks of 4 samples in early S - phase 2 samples in G2 - phase , using only common peaks . ( D ) of co - occupancy of SMC3 in early S - phase in G2 - phase with BrdU incorporation in early S - phase .

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(A) Examples of sororin (dotted arrows) and SMC3ChIP-seq and BrdU DIP-seq data from early S-phase as compared to G2-phase using the Integrated Genome Browser (IGB; Nicol et al, 2009).(B) Alignment of sororin, BrdU and BrdU-negative control sequencing bins in early S-phase to humanchromosome 18 (GRCh37/hg19). Bars correspond to called peaks, curves to read enrichments after smoothing (k=25 kbp).(C) Quantification of the co-occupancy of BrdU incorporation and sororin localization in early S-phase and sororin in G2-phase depicted as p-value distributions calculated with IntervalStats (Chikina Troyanskaya, 2012) and compared to randomized controls. Sororin peaks were identified as overlapping peaks of 4 samples in early S-phase and 2 samples in G2-phase, using only the common peaks.(D) Quantification of the co-occupancy of SMC3 in early S-phase and in G2-phase with BrdU incorporation in early S-phase.


( A ) Schematic representation of wild - type ( wt ) , targeted ( flx ) deleted ( Δ ) Cdca5 alleles ( left ) Southern blot of mouse genomic ( right ) . Red triangles , loxP sites .

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(A) Schematic representation of wild-type (wt), targeted (flx) and deleted (Δ) Cdca5 alleles (left) and Southern blot of mouse genomic DNA (right). Red triangles, loxP sites.


( B ) Observed expected genotype distribution among offspring of heterozygous Cdca5 Δ / + mice ( newborn mice , n = 136 ; E9 . 5 , n = 54 ) .

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(B) Observed and expected genotype distribution among offspring of heterozygous Cdca5 Δ/+ mice (newborn mice, n = 136; E9.5, n = 54).


( D ) Western blot of extracts to analyze depletion of sororin from fibroblasts after tamoxifen - induced Cre expression . Extracts were prepared either one or two days after release from arrest as indicated . Chr , fractionated ; sol , soluble .

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(D) Western blot of cell extracts to analyze depletion of sororin protein from fibroblasts after tamoxifen-induced Cre expression. Extracts were prepared either one or two days after release from the arrest as indicated. Chr, fractionated chromatin; sol, soluble proteins.


( E ) Proliferation curve of with indicated genotype after tamoxifen treatment .

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(E) Proliferation curve of cells with the indicated genotype after tamoxifen treatment.