( f , g ) Electroporation of GFP at E13 . 5 to E16 . 5 in TCF20 KO mice impairs distribution . n = 3 brains . Scalebar 25 μm Data information : Error bars represent means ± S . E . M . Two unpaired T - were used to analyze data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Electroporation of GFP at E13.5 to E16.5 in TCF20 KO mice impairs cell distribution. n=3 brains. Scalebar 25 μm Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**).


( h ) Chip - qPCR of TCF20 binding to TDG promoter . n = 4 biological repeats . Data information : Error bars represent means ± S . E . M . Two unpaired T - were used to analyze data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) .

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

(h) Chip-qPCR of TCF20 binding to the TDG promoter. n= 4 biological repeats. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**).


( b ) TDG ChIP - qPCR revealed that TDG mainly binds to TCF - 4 CpG islands . Seven regions ( from - 5 kbp to - 200 bp ) of promoter were analyzed . - 500 bp - 200 bp regions were located in CpG island of TCF - 4 promoter . ( n = 4 biological repeats ) . Data information : Error bars represent means ± S . E . M . Two unpaired T - were used to analyze data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) . p < 0 . 001 ( * * * ) .

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

(b) TDG ChIP-qPCR revealed that TDG mainly binds to TCF-4 CpG islands. Seven regions (from -5 kbp to -200 bp) of the promoter were analyzed. -500 bp and -200 bp regions were located in the CpG island of the TCF-4 promoter. (n=4 biological repeats). Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**). and p<0.001(***).


( c - f ) MeDIP - qPCR showed that 5fC , 5caC , 5mC are increased in TCF - 4 promoter , while 5hmC is decreased when TCF20 is insufficient . MeDIP measure region - 500 bp from TCF - 4 promoter . ( n = 4 biological repeats ) Data information : Error bars represent means ± S . E . M . Two unpaired T - were used to analyze data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) . p < 0 . 001 ( * * * ) .

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

(c-f) MeDIP-qPCR showed that 5fC, 5caC, and 5mC are increased in the TCF-4 promoter, while 5hmC is decreased when TCF20 is insufficient. The MeDIP assay measure the region -500 bp from the TCF-4 promoter. (n=4 biological repeats) Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**). and p<0.001(***).


( g ) NSCs were isolated from C57B6 / J E13 . 5 embryonic brains then were infected with a lentivirus containing a TCF20 shRNA or with a control plasmids . Immunostaining of 5fC shows that TCF20 depletion increased 5fC . dashed lines show infected with lentivirus . Scalebar 25 μm ( h ) Statistical of 5fC level . n = 20 , P < 0 . 0001 Data information : Error bars represent means ± S . E . M . Two unpaired T - were used to analyze data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) . p < 0 . 001 ( * * * ) .

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 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 O O O O O O O O O

(g) NSCs were isolated from C57B6/J E13.5 embryonic brains and then were infected with a lentivirus containing a TCF20 shRNA or with a control plasmids. Immunostaining of 5fC shows that TCF20 depletion increased 5fC levels. White dashed lines show the cells infected with lentivirus. Scalebar 25 μm (h)Statistical analysis of 5fC fluorescence level. n=20 cells, and P<0.0001 Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**). and p<0.001(***).


( i ) Immunostaining of 5caC shows that TCF20 depletion increased 5caC level . dashed lines show infected with lentivirus . Scalebar 25 μm . ( j ) Statistical of 5caC level . n = 20 , P < 0 . 0001 . Data information : Error bars represent means ± S . E . M . Two unpaired T - were used to analyze data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) . p < 0 . 001 ( * * * ) .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(i) Immunostaining of 5caC shows that TCF20 depletion increased 5caC level. White dashed lines show the cells infected with lentivirus. Scalebar 25 μm. (j) Statistical analysis of the 5caC fluorescence level. n=20 cells, and P<0.0001. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**). and p<0.001(***).


( k , l ) Overexpression of TDG TCF - 4 rescued TCF20 knockdown phenotype in brains ; n = 3 brains . Scalebar 50 μm Data information : Error bars represent means ± S . E . M . Two unpaired T - were used to analyze data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) . p < 0 . 001 ( * * * ) .

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

(k, l) Overexpression of TDG and TCF-4 rescued the TCF20 knockdown phenotype in brains; n=3 brains. Scalebar 50 μm Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**). and p<0.001(***).


B Yeast two - hybrid for interaction between above - shown Rabaptin5 segments ( Rbpt5 # , fused to LexA on bait plasmid ) residues 257 - 444 of FIP200 ( FIP , fused to Gal4 activation domain on prey plasmid ) to drive HIS3 expression . Three different clones each were replica - plated on medium with His or without His , but containing 3 - - 1 , 2 , 4 - triazole ( 3AT ; an inhibitor of His synthesis to increase stringency ) grown in absence of Trp leucine as a control . As negative controls , empty bait or prey plasmids were used . asterisk indicates a clone invalidated by recombination .

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 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 O O O O O 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 Yeast two-hybrid analysis for interaction between the above-shown Rabaptin5 segments (Rbpt5#, fused to LexA on the bait plasmid) and residues 257-444 of FIP200 (FIP, fused to the Gal4 activation domain on the prey plasmid) to drive HIS3 expression. Three different clones each were replica-plated on medium with His or without His, but containing 3-amino-1,2,4-triazole (3AT; an inhibitor of His synthesis to increase stringency) and grown in the absence of Trp and leucine as a control. As negative controls, empty bait or prey plasmids were used. The asterisk indicates a clone invalidated by recombination.


D FIP200 was immunoprecipitated ( IP ) from lysates of HeLa or HEK293A probed for FIP200 , Rabaptin5 ( Rbpt5 ) , EEA1 ( early endosome antigen 1 ) by immunoblotting . Input lysate ( 10 % ) was immunoblotted blotted parallel . As a negative control , immunoprecipitation was performed using an anti - GAPDH antibody . E - H Lysates of HeLa transiently transfected with full - FIP200 - mCherry ( FIP200 - mCh ) or a deletion mutant lacking segment interacting with Rabaptin5 ( ∆ 280 - 440 ) were immuprecipitated with anti - mCherry ( IP FIP200 - mCh ) or , as a control , with anti - FLAG antibodies ( IP FLAG ) . Immunoprecipitates input lysates ( 10 % ) were immunoblotted for mCherry Rabaptin5 ( E ) , ATG13 ( F ) , or ULK1 ( G ) . Co - immunoprecipitation of Rabaptin5 , ATG13 , ULK1 with FIP200 ∆ 280 - 440 ( FIP ∆ ) was quantified in comparison to that with wild - type FIP200 ( H ; signals normalized to that of immunoprecipitated ;

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

D FIP200 was immunoprecipitated (IP) from lysates of HeLa or HEK293A cells and probed for FIP200, Rabaptin5 (Rbpt5), and EEA1 (early endosome antigen 1) by immunoblotting. Input lysate (10%) was immunoblotted blotted parallel. As a negative control, the immunoprecipitation was performed using an anti-GAPDH antibody. E-H Lysates of HeLa cells transiently transfected with full-length FIP200-mCherry (FIP200-mCh) or a deletion mutant lacking the segment interacting with Rabaptin5 (∆280-440) were immuprecipitated with anti-mCherry (IP FIP200-mCh) or, as a control, with anti-FLAG antibodies (IP FLAG). Immunoprecipitates and input lysates (10%) were immunoblotted for mCherry and Rabaptin5 (E), ATG13 (F), or ULK1 (G). Co-immunoprecipitation of Rabaptin5, ATG13, and ULK1 with FIP200∆280-440 (FIP∆) was quantified in comparison to that with wild-type FIP200 (H; signals normalized to that of the immunoprecipitated protein;


To more easily visualize Rabaptin5 , a stable HEK293A overexpressing Rabaptin5 ( HEK + Rbpt5 ) was generated . Immunofluorescence microscopy of Rabaptin5 transferrin receptor ( TfR ) showed swelling of early endosomes upon treatment with 60 µM chloroquine for 30 min ( + CQ , panel A ) compared to untreated ( - CQ , A '

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

To more easily visualize Rabaptin5, a stable HEK293A cell line overexpressing Rabaptin5 (HEK+Rbpt5) was generated. Immunofluorescence microscopy of Rabaptin5 and transferrin receptor (TfR) showed swelling of early endosomes upon treatment with 60 µM chloroquine for 30 min (+CQ, panel A) compared to untreated cells (-CQ, A'


HEK + Rbpt5 , untransfected or 24 h after transfection with mCherry - galectin3 ( mCh - Gal3 ) , mRuby3 - galectin8 ( mRuby - Gal8 ) , mCherry - FIP200 , or - ATG16L1 were analyzed upon chloroquine treatment by immunofluorescence microscopy for Rabaptin5 mCherry - galectin3 or mRuby3 - galectin8 ( C ) , ubiquitin ( Ub ; D ) , p62 ( E ) , mCherry - FIP200 ( F ) , WIPI2 ( G ) , mCherry - ATG16L1 ( H ) , or LC3B ( I ) . Scale bar , 10 µm . In enlarged insets , arrowheads point out chloroquine - induced enlarged , ring - like early endosomes . Rabaptin5 - positive enlarged endosomes positive for mCherry - galectin3 ( Gal3 ) or mRuby3 - galectin8 ( Gal8 ) were quantified ( C ' ; mean ± S . D .

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

HEK+Rbpt5 cells, untransfected or 24 h after transfection with mCherry-galectin3 (mCh-Gal3), mRuby3-galectin8 (mRuby-Gal8), mCherry-FIP200, or -ATG16L1 were analyzed upon chloroquine treatment by immunofluorescence microscopy for Rabaptin5 and mCherry-galectin3 or mRuby3-galectin8 (C), ubiquitin (Ub; D), p62 (E), mCherry-FIP200 (F), WIPI2 (G), mCherry-ATG16L1 (H), or LC3B (I). Scale bar, 10 µm. In the enlarged insets, arrowheads point out chloroquine-induced enlarged, ring-like early endosomes. Rabaptin5-positive enlarged endosomes positive for mCherry-galectin3 (Gal3) or mRuby3-galectin8 (Gal8) were quantified (C'; mean ± S.D.


J , K HEK + Rbpt5 , untransfected or 24 h after transfection with mCherry - ATG16L1 , were treated with 60 µM chloroquine for 0 , 15 30 min stained for Rabaptin5 either WIPI2 or mCherry - ATG16L1 . Manders ' colocalization coefficients were determined , M1 showing fraction of Rabaptin5 - positive structures also positive for WIPI2 ( J ) or mCherry - ATG16L1 ( K ) , M2 showing respective inverse ( mean ± S . D . of three independent experiments ;

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

J, K HEK+Rbpt5 cells, untransfected or 24 h after transfection with mCherry-ATG16L1, were treated with 60 µM chloroquine for 0, 15 and 30 min and stained for Rabaptin5 and either WIPI2 or mCherry-ATG16L1. Manders' colocalization coefficients were determined, M1 showing the fraction of Rabaptin5-positive structures also positive for WIPI2 (J) or mCherry-ATG16L1 (K), and M2 showing the respective inverse (mean ± S.D. of three independent experiments;


A HEK293A were transfected with nontargeting siRNA ( siCtr ) or siRNAs silencing Rabaptin5 ( siRbpt5 ) or FIP200 ( siFIP200 ) for 72 h treated without ( - ) or with 60 µM chloroquine ( + CQ ) or 250 nM Torin1 for 150 min . Scale bar , 10 µm . Below , efficiency of Rabaptin5 FIP200 knockdown was assayed by immunoblotting using tubulin ( Tub ) as a loading control . B WIPI2 or LC3B per were quantified for each condition ( mean ± S . D . of three independent experiments ; ANOVA : * P < 0 . 05 , * * P < 0 . 01 ) .

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

A HEK293A cells were transfected with nontargeting siRNA (siCtr) or siRNAs silencing Rabaptin5 (siRbpt5) or FIP200 (siFIP200) for 72 h and treated without (-) or with 60 µM chloroquine (+CQ) or 250 nM Torin1 for 150 min. Scale bar, 10 µm. Below, the efficiency of Rabaptin5 and FIP200 knockdown was assayed by immunoblotting using tubulin (Tub) as a loading control. B WIPI2 or LC3B puncta per cell were quantified for each condition (mean ± S.D. of three independent experiments; ANOVA: *P<0.05, **P<0.01). C


C HEK293A were transfected with siCtr or siRbpt5 as in A incubated without or with 280 µM LLOMe for 150 min to induce lysophagy . were fixed immunostained for endogenous WIPI2 LC3B . Scale bar , 10 µm . D WIPI2 or LC3B per were quantified ( mean ± S . D . of three independent experiments ) .

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C HEK293A cells were transfected with siCtr or siRbpt5 as in A and incubated without or with 280 µM LLOMe for 150 min to induce lysophagy. Cells were fixed and immunostained for endogenous WIPI2 and LC3B. Scale bar, 10 µm. D WIPI2 or LC3B puncta per cell were quantified (mean ± S.D. of three independent experiments).


A HEK293A were transfected with nontargeting siRNA ( siCtr ) or siRNAs silencing ATG13 ( siATG13 ) for 72 h , treated without ( - ) or with 60 µM chloroquine ( + CQ ) for 150 minutes , fixed immunostained for endogenous WIPI2 LC3B . Scale bar , 10 µm . Efficiency of ATG13 knockdown was assayed by immunoblotting below on right using GAPDH as loading controls . B WIPI2 of LC3B per were quantified for each condition ( mean ± S . D . of three independent experiments ; ANOVA : * * P < 0 . 01 ) .

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 B-MEASURED_VAR I-MEASURED_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 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 O O O O O O O O O O O O

A HEK293A cells were transfected with nontargeting siRNA (siCtr) or siRNAs silencing ATG13 (siATG13) for 72 h, treated without (-) or with 60 µM chloroquine (+CQ) for 150 minutes, and fixed and immunostained for endogenous WIPI2 and LC3B. Scale bar, 10 µm. Efficiency of ATG13 knockdown was assayed by immunoblotting below on the right using GAPDH as loading controls. B WIPI2 of LC3B puncta per cell were quantified for each condition (mean ± S.D. of three independent experiments; ANOVA: **P<0.01). C


HEK293A were transfected with nontargeting siRNA ( siCtr ) or siRNAs silencing ULK1 ( siULK1 ) , ULK2 ( siULK2 ) , or both ( siULK1 + 2 ) for 72 h , treated without ( - ) or with 60 µM chloroquine ( + CQ ) for 150 minutes , fixed immunostained for endogenous WIPI2 LC3B . Scale bar , 10 µm . Efficiency of ULK1 knockdown was assayed by immunoblotting efficiency of ULK2 knockdown was assayed by quantitative RT - PCR result is shown in panel C D WIPI2 of LC3B per were quantified for each condition ( mean ± S . D . of three independent

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

HEK293A cells were transfected with nontargeting siRNA (siCtr) or siRNAs silencing ULK1 (siULK1), ULK2 (siULK2), or both (siULK1+2) for 72 h, treated without (-) or with 60 µM chloroquine (+CQ) for 150 minutes, and fixed and immunostained for endogenous WIPI2 and LC3B. Scale bar, 10 µm. Efficiency of ULK1 knockdown was assayed by immunoblotting The efficiency of ULK2 knockdown was assayed by quantitative RT-PCR and the result is shown in panel C D WIPI2 of LC3B puncta per cell were quantified for each condition (mean ± S.D. of three independent experiments


E HEK293A were incubated without ( - ) , with 60 µM chloroquine ( + CQ ) , or with chloroquine 5 µM MRT68921 ( + CQ + MRT ) for 150 min fixed stained for WIPI2 LC3B . F WIPI2 of LC3B per were quantified for each condition as in panel B ( mean ± S . D . of three independent experiments ) .

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E HEK293A cells were incubated without (-), with 60 µM chloroquine (+CQ), or with chloroquine and 5 µM MRT68921 (+CQ +MRT) for 150 min and fixed and stained for WIPI2 and LC3B. F WIPI2 of LC3B puncta per cell were quantified for each condition as in panel B (mean ± S.D. of three independent experiments).


B Co - immunoprecipitation was performed as in panel A using parental HEK293A CRISPR / Cas9 knockout lacking FIP200 ( FIP - KO ) . Anti - HA antibodies were used as a control ( IP HA ) . On right , HEK293A FIP200 knockout were immunoblotted for FIP200 as a loading control of tubulin ( Tub ) . Signals were quantified ratios of mCherry - ATG16L1 / Rabaptin5 normalized to that of HEK293A without chloroquine

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 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-MEASURED_VAR O 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

B Co-immunoprecipitation was performed as in panel A using parental HEK293A cells and CRISPR/Cas9 knockout cells lacking FIP200 (FIP-KO). Anti-HA antibodies were used as a control (IP HA). On the right, HEK293A and FIP200 knockout cells were immunoblotted for FIP200 and as a loading control of tubulin (Tub). Signals were quantified and the ratios of mCherry-ATG16L1/Rabaptin5 normalized to that of HEK293A cells without chloroquine treatment


Lysates of HEK293A or HeLa transiently transfected with full - mCherry - ATG16L1 ( wt ) or a mutant lacking WD domain ( ∆ WD ; residues 1 - 319 of ATG16L1 , precisely deleting only WD40 repeats residues 320 - 607 ) were immunoprecipitated with anti - Rabaptin5 or anti - FLAG antibodies , immunoblotted for Rabaptin5 mCherry - ATG16L1 , or immunoprecipitated with anti - FIP200 or anti - HA antibodies Co - immunoprecipitation of ATG16L1 ∆ WD with Rabaptin5 ( C ) was reduced to 6 . 6 ± 2 . 1 % 3 . 4 ± 2 . 1 % in HEK293A HeLa , respectively , relative to that of full - ATG16L1 ( signals normalized to immunoprecipitated ; mean ± S . D . deviation of three independent experiments each ) .

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

Lysates of HEK293A or HeLa cells transiently transfected with full-length mCherry-ATG16L1 (wt) or a mutant lacking the WD domain (∆WD; residues 1-319 of ATG16L1, precisely deleting only the WD40 repeats residues 320-607) were immunoprecipitated with anti-Rabaptin5 or anti-FLAG antibodies, and immunoblotted for Rabaptin5 and mCherry-ATG16L1 , or immunoprecipitated with anti-FIP200 or anti-HA antibodies Co-immunoprecipitation of ATG16L1∆WD with Rabaptin5 (C) was reduced to 6.6±2.1% and 3.4±2.1% in HEK293A and HeLa cells, respectively, relative to that of full-length ATG16L1 (signals normalized to the immunoprecipitated protein; mean ± S.D. deviation of three independent experiments each).


F Lysates of HeLa transiently transfected with myc - tagged wild - type Rabaptin5 ( wt ) or triple - alanine mutant ( AAA ) were immunoprecipitated with anti - myc ( IP myc ) or anti - FLAG antibodies ( IP FLAG ) , immunoblotted for myc ATG16L1 . Co - immunoprecipitation of ATG16L1 with Rabaptin5 - AAA triple mutant was reduced to 1 . 5 ± 1 . 2 % relative to that with wild - type Rabaptin5 ( signals normalized to that of immunoprecipitated ; mean ± S . D . of three independent experiments ) .

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F Lysates of HeLa cells transiently transfected with myc-tagged wild-type Rabaptin5 (wt) or triple-alanine mutant (AAA) were immunoprecipitated with anti-myc (IP myc) or anti-FLAG antibodies (IP FLAG), and immunoblotted for myc and ATG16L1. Co-immunoprecipitation of ATG16L1 with Rabaptin5-AAA triple mutant was reduced to 1.5±1.2% relative to that with wild-type Rabaptin5 (signals normalized to that of the immunoprecipitated protein; mean ± S.D. of three independent experiments).


A By immunoblot , of Rabaptin5 as a loading control of tubulin ( Tub ) were assessed in wild - type HEK293A , HEK + Rbpt5 stably overexpressing Rabaptin5 , Rabaptin5 - knockout without ( Rbpt5 - KO ) or with stable re - expression of wild - type ( Rbpt5 - KO + wt ) or AAA - mutant Rabaptin5 ( Rbpt5 - KO + AAA ) . B same stable HEK293A - derived lines were treated without ( - CQ ) or with 60 µM chloroquine for 150 min ( + CQ ) analyzed by immunofluorescence microscopy for WIPI2 or LC3B . Scale bar , 10 µm .

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A By immunoblot analysis, the levels of Rabaptin5 and as a loading control of tubulin (Tub) were assessed in wild-type HEK293A cells, HEK+Rbpt5 cells stably overexpressing Rabaptin5, and Rabaptin5-knockout cells without (Rbpt5-KO) or with stable re-expression of wild-type (Rbpt5-KO+wt) or AAA-mutant Rabaptin5 (Rbpt5-KO+AAA). B The same stable HEK293A-derived cell lines were treated without (-CQ) or with 60 µM chloroquine for 150 min (+CQ) and analyzed by immunofluorescence microscopy for WIPI2 or LC3B. Scale bar, 10 µm. C


D , E HEK293A - derived lines were treated with 60 µM chloroquine for 30min non - lipidated lipidated LC3B ( I II , resp . ) were assayed by immunoblot ( D ) . increase of LC3B - II fraction of total LC3B upon chloroquine treatment was quantified ( mean ± S . D . of three independent experiments ;

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D, E The HEK293A-derived cell lines were treated with 60 µM chloroquine for 30min and non-lipidated and lipidated LC3B (I and II, resp.) were assayed by immunoblot analysis (D). The increase of the LC3B-II fraction of total LC3B upon chloroquine treatment was quantified (mean ± S.D. of three independent experiments;


F , G HEK + Rbpt5 Rabaptin5 - knockout stably re - expressing wild - type ( Rbpt5 - KO + wt ) or AAA - mutant Rabaptin5 ( Rbpt5 - KO + AAA ) were transfected with mCherry - ATG16L1 , treated with 60 µM chloroquine ( CQ ) for 0 , 15 30 min , analyzed by immunofluorescence microscopy for Rabaptin5 , mCherry - ATG16L1 , WIPI2 . Manders ' colocalization coefficients were determined , M1 showing fraction of Rabaptin5 - positive structures also positive for mCherry - ATG16L1 ( F ) or WIPI2 ( G ) M2 showing inverse ( mean ± S . D . of three independent

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F, G HEK+Rbpt5 cells and Rabaptin5-knockout cells stably re-expressing wild-type (Rbpt5-KO+wt) or AAA-mutant Rabaptin5 (Rbpt5-KO+AAA) were transfected with mCherry-ATG16L1, treated with 60 µM chloroquine (CQ) for 0, 15 and 30 min, and analyzed by immunofluorescence microscopy for Rabaptin5, mCherry-ATG16L1, and WIPI2. Manders' colocalization coefficients were determined, M1 showing the fraction of Rabaptin5-positive structures also positive for mCherry-ATG16L1 (F) or WIPI2 (G) and M2 showing the inverse (mean ± S.D. of three independent experiments


H Wild - type HEK293A , HEK + Rbpt5 , Rabaptin5 - knockout without ( Rbpt5 - KO ) or with stable re - expression of wild - type ( Rbpt5 - KO + wt ) or AAA - mutant Rabaptin5 ( Rbpt5 - KO + AAA ) were stained with lysotracker with DAPI for nuclei . Scale bar , 10 µm . I number of lysosomes ( lysotracker - positive structures ) per was quantified from as in panel C ( mean ± S . D . of three independent experiments ) . for at least 15 per sample were quantified . value for HEK + Rbpt5 is an underestimate , because density of makes them difficult to distinguish as separate structures .

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H Wild-type HEK293A cells, HEK+Rbpt5 cells, Rabaptin5-knockout cells without (Rbpt5-KO) or with stable re-expression of wild-type (Rbpt5-KO+wt) or AAA-mutant Rabaptin5 (Rbpt5-KO+AAA) were stained with lysotracker and with DAPI for nuclei. Scale bar, 10 µm. I The number of lysosomes (lysotracker-positive structures) per cell was quantified from cells as in panel C (mean ± S.D. of three independent experiments). Images for at least 15 cells per sample were quantified. The value for HEK+Rbpt5 cells is an underestimate, because the density of puncta makes them difficult to distinguish as separate structures.


A HeLa were transfected with nontargeting siRNA ( siCtr ) or siRNAs silencing Rabaptin5 ( siRbpt5 ) or FIP200 ( siFIP200 ) for 72 h . were infected with Salmonella by centrifugation at 500 × g for 5min at 37 ° C incubation for 10 min at 37 ° C , washed three times , incubated in fresh culture medium containing gentamicin to prevent growth of extracellular for 0 , 1 , 3 , or 6 h before lysis of host plating of on LB agar plates at various dilutions to determine number of live at different time points , shown as a of internalized after infection ( mean ± S . D . of three independent experiments ) . fractions of internalized alive 1 h after infection are shown separately in middle ( mean ± S . D . of three independent experiments ; ANOVA : * P < 0 . 05 ) . On right , fraction of infected was determined for HeLa transfected with siRNAs infected as above with Salmonella expressing GFP , washed , immediately fixed for microscopy stained with anti - transferrin receptor anti - LC3B as markers . Z - stacks for > 5 ' 000cells / sample were acquired analyzed in Fiji to determine fraction of infected ( mean ± S . D . of three independent experiments ) . average number of per infected was identical ( 2 . 16 , 2 . 11 , 2 . 12 per transfected with siCtr , siRbpt5 , siFIP200 , respectively ) .

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A HeLa cells were transfected with nontargeting siRNA (siCtr) or siRNAs silencing Rabaptin5 (siRbpt5) or FIP200 (siFIP200) for 72 h. The cells were infected with Salmonella by centrifugation at 500×g for 5min at 37°C and incubation for 10 min at 37°C, washed three times, and incubated in fresh culture medium containing gentamicin to prevent growth of extracellular bacteria for 0, 1, 3, or 6 h before lysis of the host cells and plating of the bacteria on LB agar plates at various dilutions to determine the number of live bacteria at the different time points, shown as a percentage of internalized cells after infection (mean ± S.D. of three independent experiments). The fractions of internalized bacteria alive 1 h after infection are shown separately in the middle (mean ± S.D. of three independent experiments; ANOVA: *P<0.05). On the right, the fraction of infected cells was determined for HeLa cells transfected with siRNAs and infected as above with Salmonella expressing GFP, washed, and immediately fixed for fluorescence microscopy and stained with anti-transferrin receptor and anti-LC3B as cellular markers. Z-stacks for >5'000cells/sample were acquired and analyzed in Fiji to determine the fraction of infected cells (mean ± S.D. of three independent experiments). The average number of bacteria per infected cell was identical (2.16, 2.11, and 2.12 bacteria per cell transfected with siCtr, siRbpt5, and siFIP200, respectively).


B Wild - type HEK293A , HEK + Rbpt5 , Rbpt5 - KO , Rbpt5 - KO + wt , Rbpt5 - KO + AAA , FIP200 - KO were infected with Salmonella treated analyzed as in panels A ( mean ± S . D . of three independent experiments ; ANOVA : * P < 0 . 05 , * * * * P < 0 . 0001 ) .

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B Wild-type HEK293A, HEK+Rbpt5, Rbpt5-KO, Rbpt5-KO+wt, Rbpt5-KO+AAA, and FIP200-KO cells were infected with Salmonella and treated and analyzed as in panels A (mean ± S.D. of three independent experiments; ANOVA: *P<0.05, ****P<0.0001).


C Wild - type HEK293A , HEK + Rbpt5 , Rbpt5 - KO , Rbpt5 - KO + AAA , FIP200 - KO were infected with Salmonella expressing GFP as in panel B , incubated in fresh culture medium containing gentamicin for 0 , 5 , 15 , 30 , 60 min , fixed with methanol immunostained for transferrin receptor ( TfR ) as a marker of early endosomes for LC3B as a marker of autophagy . Salmonella were classified according to their association with a TfR - / or LC3B - positive compartment - as illustrated on top left ( scale bar , 2 µm ) - during first hour after infection . In absence of Rabaptin5 , LC3 - positive SCVs with early endosomal characteristics ( containing TfR ) were strongly reduced . ( Mean ± S . D . of three independent experiments , analyzing > 50 for each time point . )

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C Wild-type HEK293A, HEK+Rbpt5, Rbpt5-KO, Rbpt5-KO+AAA, and FIP200-KO cells were infected with Salmonella expressing GFP as in panel B, incubated in fresh culture medium containing gentamicin for 0, 5, 15, 30, and 60 min, fixed with methanol and immunostained for transferrin receptor (TfR) as a marker of early endosomes and for LC3B as a marker of autophagy. Salmonella were classified according to their association with a TfR- and/or LC3B-positive compartment - as illustrated on the top left (scale bar, 2 µm) - during the first hour after infection. In the absence of Rabaptin5, LC3-positive SCVs with early endosomal characteristics (containing TfR) were strongly reduced. (Mean ± S.D. of three independent experiments, analyzing >50 bacteria for each time point.)


B : Consequences of ROS on SUMO proteome vary amongst lines . HeLa , MCF7 , U2OS or RPE were treated with increasing H2O2 for 15 min lysed in Laemmli buffer supplemented with 20 mM N - Ethylmaleimide ( NEM ) . Samples were analyzed by immunoblotting with SUMO1 antibodies .

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B: Consequences of ROS on the SUMO proteome vary amongst cell lines. HeLa, MCF7, U2OS or RPE cells were treated with increasing H2O2 concentration for 15 min and lysed in Laemmli buffer supplemented with 20 mM N-Ethylmaleimide (NEM). Samples were analyzed by immunoblotting with SUMO1 antibodies.


C : H2O2 induces loss of TRIM28 SUMOylation in HeLa but not in U2OS . HeLa or U2OS were mock treated or treated with 500 µM H2O2 for 5 or 15 min . were lysed with 1 % SDS in PBS buffer 20 mM NEM . Upon immunoprecipitation of SUMO1 proteome , samples were analyzed by immunoblotting with TRIM28 antibodies .

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C: H2O2 induces loss of TRIM28 SUMOylation in HeLa cells but not in U2OS cells. HeLa or U2OS cells were mock treated or treated with 500 µM H2O2 for 5 or 15 min. Cells were lysed with 1% SDS in PBS buffer and 20 mM NEM. Upon immunoprecipitation of the SUMO1 proteome, samples were analyzed by immunoblotting with TRIM28 antibodies.


D : Induction of Uba2 ~ Ubc9 disulfide is a conserved mechanism . HeLa , MCF7 , U2OS or RPE were treated with increasing of H2O2 for 15 min , lysed in non reducing buffer supplemented with 20 mM NEM , analyzed by immunoblotting with Uba2 antibodies .

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D: Induction of the Uba2~Ubc9 disulfide is a conserved mechanism. HeLa, MCF7, U2OS or RPE cells were treated with increasing concentration of H2O2 for 15 min, lysed in non reducing buffer supplemented with 20 mM NEM, and analyzed by immunoblotting with Uba2 antibodies.


A : Upper panel : Strategy for identification of oxidation resistant Ubc9 variants . Ubc9 was mutagenized by PCR under conditions that introduced 1 - 3 random mutations . Ubc9 variants were expressed in lysates of clones tested for E2 activity in presence or absence of 1 mM H2O2 . For this purpose , we used a FRET - in vitro SUMOylation with recombinant SUMO E1 , YFP - SUMO CFP - RanGAPtail ATP . Lower panel : Ubc9 W103R is H2O2 resistant . lysates containing Ubc9 wt ( left panel ) or Ubc9 W103R ( right panel ) were tested as described .

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A: Upper panel: Strategy for the identification of oxidation resistant Ubc9 variants. Ubc9 was mutagenized by PCR under conditions that introduced 1 - 3 random mutations. Ubc9 variants were expressed in bacteria and the bacterial lysates of individual clones tested for E2 activity in the presence or absence of 1 mM H2O2. For this purpose, we used a FRET-based in vitro SUMOylation assay with recombinant SUMO E1, YFP-SUMO and CFP-RanGAPtail and ATP. Lower panel: Ubc9 W103R is H2O2 resistant. Bacterial lysates containing Ubc9 wt (left panel) or Ubc9 W103R (right panel) were tested as described.


B : Ubc9 W103 mutants are H2O2 resistant but not fully active . Left panel : Recombinant Ubc9 W103R , W103A W103F were purified ( embedded panel ) . Resistance against oxidation was tested under conditions of limiting E1 enzyme : 21 nM Aos1 / Uba2 73 nM Ubc9 were incubated with H2O2 prior to addition of 160 nM each of YFP - SUMO1 CFP - RanGAPtail . Right panel : To compare specific activities of wt mutants in absence of H2O2 , sumoylation assays were carried out using limiting Ubc9 . Reactions contained 35 nM Aos1 / Uba2 , 11 nM Ubc9 , 85 nM each of YFP - SUMO1 CFP - RanGAPtail 1 mM ATP .

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B: Ubc9 W103 mutants are H2O2 resistant but not fully active. Left panel: Recombinant Ubc9 W103R, W103A and W103F were purified (embedded panel). Resistance against oxidation was tested under conditions of limiting E1 enzyme: 21 nM Aos1/Uba2 and 73 nM Ubc9 were incubated with H2O2 prior to the addition of 160 nM each of YFP-SUMO1 and CFP-RanGAPtail. Right panel: To compare specific activities of wt and mutants in the absence of H2O2, sumoylation assays were carried out using limiting Ubc9 concentration. Reactions contained 35 nM Aos1/Uba2, 11 nM Ubc9, 85 nM each of YFP-SUMO1 and CFP-RanGAPtail and 1 mM ATP.


B : In FRET - , Ubc9 D100A is active upon H2O2 treatment . Ubc9 D100A was purified compared for activity in presence of H2O2 as described in Fig . 2B .

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B: In FRET-based assay, Ubc9 D100A is active upon H2O2 treatment. Ubc9 D100A was purified and compared for activity in the presence of H2O2 as described in Fig. 2B.


C : Ubc9 wt D100A are equally active in CFP - RanGAPtail SUMOylation . Assays were with 35 nM Aos1 / Uba2 15 nM Ubc9 .

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C: Ubc9 wt and D100A are equally active in CFP-RanGAPtail SUMOylation. Assays were with 35 nM Aos1/Uba2 and 15 nM Ubc9.


D : Ubc9 D100A is fully competent to form a thioester with SUMO . Single turnover reactions were performed using 630 nM E1 , 3 , 3 µM SUMO1 600 nM Ubc9 . Reactions were stopped at different time points by addition of non - reducing buffer . .

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D: Ubc9 D100A is fully competent to form a thioester with SUMO. Single turnover reactions were performed using 630 nM E1, 3,3 µM SUMO1 and 600 nM Ubc9. Reactions were stopped at different time points by addition of non-reducing buffer..


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E: Ubc9 wt and D100A are equally active in PIAS2b - dependent SUMOylation. Assays were with 300 nM GST-p53, 170 nM E1, 120 nM Ubc9, 5 µM SUMO2 and 49 nM GST-Pias2b


F : Ubc9 wt or D100A are equally active in RanBP2 - dependent SUMOylation . Assays were with 650 nM YFP - SP100 , 110 nM of E1 , 25 Ubc9 , 40 nM RanBP2ΔFG 3 , 5 µM SUMO1 ( Left panel ) or SUMO2 ( Right panel ) .

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F: Ubc9 wt or D100A are equally active in RanBP2 - dependent SUMOylation. Assays were with 650 nM YFP-SP100, 110 nM of E1, 25 Ubc9, 40 nM RanBP2ΔFG and 3,5 µM SUMO1 (Left panel) or SUMO2 (Right panel).


A : Ubc9 D100A enzymatic activity can be recovered by limited amount of DTT . 21 nM Aos1 / Uba2 75 nM Ubc9 were incubated with H2O2 for 30 min prior addition of 65 nM each YFP - SUMO1 CFP - RanGAPtail diluted in DTT - free buffer . final DTT in reaction was 6 µM . Prior to addition of ATP , extra amount of DTT was added at indicated final .

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A: Ubc9 D100A enzymatic activity can be recovered by limited amount of DTT. 21 nM Aos1/Uba2 and 75 nM Ubc9 were incubated with H2O2 for 30 min prior addition of 65 nM each YFP-SUMO1 and CFP-RanGAPtail diluted in DTT-free buffer. The final DTT concentration in the reaction was 6 µM. Prior to addition of ATP, extra amount of DTT was added at indicated final concentration.


B : Both Uba2 ~ Ubc9 disulfide formation reduction are accelerated with Ubc9 D100A . 100 nM E1 1 µM E2 were incubated in presence of 1 mM H2O2 . One hour after H2O2 addition , 500 µM reduced glutathione was added . Samples were taken at indicated time points Uba2 ~ Ubc9 disulfide was monitored by immunoblotting against Uba2 . Lower panel : of between crosslinked Uba2 vs total

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B: Both Uba2~Ubc9 disulfide formation and reduction are accelerated with Ubc9 D100A.100 nM E1 and 1 µM E2 were incubated in presence of 1 mM H2O2. One hour after H2O2 addition, 500 µM reduced glutathione was added. Samples were taken at indicated time points and the Uba2~Ubc9 disulfide was monitored by immunoblotting against Uba2. Lower panel: Quantification of the ratio between crosslinked Uba2 vs total Uba2


A : Generation of polyclonal populations of stable . Top panel : Replacement strategy : U2OS transfected with pIRES - hrGFP II - vectors were selected via addition of G418 . After 4 , low level GFP expressing were sorted by FACS after 4 again after 8 . Bottom panel : Stable express Ubc9 wt D100A at comparable to endogenous Ubc9 : U2OS or U2OS stable were transfected with siRNA targeting human Ubc9 mRNA for 72h or mocked transfected . were lysed in Laemmli buffer Ubc9 a - tubulin as loading control were detected by immunoblotting .

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A: Generation of polyclonal populations of stable cells. Top panel: Replacement strategy: U2OS cells transfected with pIRES-hrGFP II-based vectors were selected via addition of G418. After 4 weeks, low level GFP expressing cells were sorted by FACS after 4 weeks and again after 8 weeks. Bottom panel: Stable cells express Ubc9 wt and D100A at levels comparable to endogenous Ubc9: U2OS or U2OS stable cells were transfected with siRNA targeting human Ubc9 mRNA for 72h or mocked transfected. Cells were lysed in Laemmli buffer and Ubc9 and a-tubulin as loading control were detected by immunoblotting.


B : Ubc9 D100A is severely impaired for disulfide bond formation with Uba2 . Stable U2OS were transfected with siRNA against Ubc9 for 72h , treated with 250 µM H2O2 for different time points , from 2 min to 2 hours , lysed in non - reducing buffer analyzed by immunoblotting against Ubc9 to detect disulfide ( upper panel ) or free Uba2 as loading control ( see also Appendix Fig . S5 ) . Lower panel : relative intensities of Ubc9 bands were measured using imageJ . Highest intensities in both population were set to 1 .

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B: Ubc9 D100A is severely impaired for disulfide bond formation with Uba2. Stable U2OS cells were transfected with siRNA against Ubc9 for 72h, treated with 250 µM H2O2 for different time points, from 2 min to 2 hours, lysed in non-reducing buffer and analyzed by immunoblotting against Ubc9 to detect the disulfide (upper panel) or free Uba2 as loading control (see also Appendix Fig. S5). Lower panel: relative intensities of Ubc9 bands were measured using imageJ. Highest intensities in both cell population were set to 1.


C : Ubc9 D100A impairs survival upon H2O2 treatment . Ubc9 wt or Ubc9 D100A were transfected with siRNA against endogenous Ubc9 for 72h , transferred into 6 well plates ( 20 000 / well ) treated with increasing H2O2 for 1h . Ten days later , were washed with PBS , fixed with 4 % formaldehyde stained with crystal violet . occupied by was evaluated using ImageJ ColonyArea plugin . An arbitrary value of 1 was given to occupied by non - treated . graph is showing mean of 4 independent experiments . Error bars are S . E . M . * : p < 0 .

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C: Ubc9 D100A impairs cell survival upon H2O2 treatment. Ubc9 wt or Ubc9 D100A cells were transfected with siRNA against endogenous Ubc9 for 72h, transferred into 6 well plates (20 000 cells / well) and treated with increasing H2O2 concentration for 1h. Ten days later, cells were washed with PBS, fixed with 4% formaldehyde and stained with crystal violet. The area occupied by the cells was evaluated using ImageJ and the ColonyArea plugin. An arbitrary value of 1 was given to the area occupied by non-treated cells. The graph is showing the mean of 4 independent experiments. Error bars are S.E.M. *: p < 0.05


D : Constitutive expression of Ubc9 D100A affects cellgrowth . 103 were plated per 10 cm plate cultivated for 10 days in 20 % or 5 % O2 incubators . Subsequently , were colored using crystal violet . of each clone was measured using ImageJ . Representative plates are shown in upper panel . Lower left panel : Distribution of colony sizes , shown for one of three biological replicates . Lower right panel : Comparison of colonies between " Ubc9 wt " " Ubc9 DA " . average of " Ubc9 wt " was set to 1 . Shown is mean of 3 independent experiments , each of which was performed in technical triplicates . Error bars are S . E . M .

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D: Constitutive expression of Ubc9 D100A affects cellgrowth. 103 cells were plated per 10 cm plate and cultivated for 10 days in 20% or 5% O2 incubators. Subsequently, cells were colored using crystal violet. The area of each clone was measured using ImageJ. Representative plates are shown in the upper panel. Lower left panel: Distribution of colony sizes, shown for one of the three biological replicates. Lower right panel: Comparison of the colonies size between "Ubc9 wt"and "Ubc9 DA" cells. The average size of "Ubc9 wt" was set to 1. Shown is the mean of 3 independent experiments, each of which was performed in technical triplicates. Error bars are S.E.M.


A : Phosphorylation of ATM is impaired in Ubc9 D100A . Stable U2OS populations were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 250 µM H2O2 . At indicated times , were lysed in Laemmli buffer analysed by immunoblotting with indicated antibodies .

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A: Phosphorylation of ATM is impaired in Ubc9 D100A cells. Stable U2OS cell populations were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 250 µM H2O2. At the indicated times, cells were lysed in Laemmli buffer and analysed by immunoblotting with the indicated antibodies.


B : Phosphorylation of gamma - H2AX is impaired in Ubc9 D100A . Experiment was performed as in A . Stable U2OS populations were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 250 µM H2O2 . At indicated times , were lysed in Laemmli buffer analysed by immunoblotting with indicated antibodies .

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B: Phosphorylation of gamma-H2AX is impaired in Ubc9 D100A. Experiment was performed as in A. Stable U2OS cell populations were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 250 µM H2O2. At the indicated times, cells were lysed in Laemmli buffer and analysed by immunoblotting with the indicated antibodies.


C : Phosphorylation of Chk2 is impaired in Ubc9 D100A . Experiment was performed as described in A . Stable U2OS populations were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 250 µM H2O2 . At indicated times , were lysed in Laemmli buffer analysed by immunoblotting with indicated antibodies .

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C: Phosphorylation of Chk2 is impaired in Ubc9 D100A. Experiment was performed as described in A. Stable U2OS cell populations were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 250 µM H2O2. At the indicated times, cells were lysed in Laemmli buffer and analysed by immunoblotting with the indicated antibodies.


D : Ubc9 D100A are fully competent for phosphorylation of ATM upon hydroxyurea exposure . Stable U2OS populations were exposed to 2 . 5 mM hydroxyurea or 250 µM H2O2 . were lysed at indicated time points analysed by immunoblotting with indicated antibodies .

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D: Ubc9 D100A cells are fully competent for phosphorylation of ATM upon hydroxyurea exposure. Stable U2OS cell populations were exposed to 2.5 mM hydroxyurea or 250 µM H2O2. Cells were lysed at indicated time points and analysed by immunoblotting with the indicated antibodies.


E : Phosphorylation of Chk1 is not impaired in Ubc9 D100A . Stable U2OS were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 2 . 5 mM hydroxyurea . At indicated times , were lysed in Laemmli buffer analysed by immunoblotting with indicated antibodies .

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E: Phosphorylation of Chk1 is not impaired in Ubc9 D100A cells. Stable U2OS cells were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 2.5 mM hydroxyurea. At the indicated times, cells were lysed in Laemmli buffer and analysed by immunoblotting with the indicated antibodies.


A : Impaired y - H2AX 53BP1 accumulation in Ubc9 D100A . Stable U2OS were transfected with siRNA against endogenous Ubc9 for 72 h . were treated with 125 µM H2O2 immunofluorescence microscopy was performed . Left panel : representative immunofluorescence . Right panel : of with γ - H2AX 53BP1 foci . Error bars represent S . E . M , n = 3 independent experiments * p < 0 . 05 .

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A: Impaired y-H2AX and 53BP1 accumulation in Ubc9 D100A cells. Stable U2OS cells were transfected with siRNA against endogenous Ubc9 for 72 h. Cells were treated with 125 µM H2O2 and immunofluorescence microscopy was performed. Left panel: representative immunofluorescence images. Right panel: quantification of cells with γ-H2AX and 53BP1 foci. Error bars represent S.E.M, n=3 independent experiments * p < 0.05.


B : H2O2 exposure leads to prolonged damage in Ubc9 wt Ubc9 D100A . were treated as described in A . Stable U2OS were transfected with siRNA against endogenous Ubc9 for 72 h . were treated with 125 µM H2O2 immunofluorescence microscopy was performed . An alkaline comet was performed to evaluate damage . Representative are shown . Error intervals represent S . E . M , n = 4 independent experiments .

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B: H2O2 exposure leads to prolonged DNA damage in Ubc9 wt and Ubc9 D100A cells. Cells were treated as described in A. Stable U2OS cells were transfected with siRNA against endogenous Ubc9 for 72 h. Cells were treated with 125 µM H2O2 and immunofluorescence microscopy was performed. An alkaline comet assay was performed to evaluate the DNA damage. Representative images are shown. Error intervals represent S.E.M, n=4 independent experiments.


A : Diamide is inducing SUMO E1 ~ E2 disulfide but not ATM phosphorylation . U2OS stable populations were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 500 µM diamide or 500 µM H2O2 . were subsequently washed in PBS with 20 mM NEM lysed in Laemmli buffer . lysates were resolved by SDS - PAGE .

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A: Diamide is inducing the SUMO E1~E2 disulfide but not ATM phosphorylation. U2OS stable cell populations were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 500 µM diamide or 500 µM H2O2. Cells were subsequently washed in PBS with 20 mM NEM and lysed in Laemmli buffer. The lysates were resolved by SDS-PAGE.


B : Left panel : PP2A inhibitor okadaic acid rescues y - H2AX defect of " Ubc9 DA " . " Ubc9 wt " or " Ubc9 DA " were treated with 0 . 25 µM okadaic acid for 30 min . After addition of 250 µM H2O2 in fresh medium for 3 hours , immunofluorescence microscopy was performed as described in Fig . 7A . Error bars represent S . E . M , n = 3 independent experiments . Right panel : Representative immunofluorescence .

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B: Left panel: The PP2A inhibitor okadaic acid rescues the y-H2AX defect of the "Ubc9 DA" cells. "Ubc9 wt" or "Ubc9 DA" cells were treated with 0.25 µM okadaic acid for 30 min. After addition of 250 µM H2O2 in fresh medium for 3 hours, immunofluorescence microscopy was performed as described in Fig. 7A. Error bars represent S.E.M, n=3 independent experiments. Right panel: Representative immunofluorescence images.


C : Left panel : Ubc9 D100A enhances sensitivity to Ara - C or etoposide . survival assays were performed as described in Fig . 5C . were exposed to Ara - C or etoposide ( VP16 ) for 1 h evaluated 10 days later . Right panel : N - acetylcysteine treatment rescues higher toxicity of Ara - C or etoposide on " Ubc9 DA " . were treated with 0 . 5 mM NAC 16 h prior to addition of etoposide or Ara - C . Graphs represent means of three to four independent experiments , error bars represent S . E . M . * p < 0 . 05 .

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C: Left panel: Ubc9 D100A enhances sensitivity to Ara-C or etoposide. Cell survival assays were performed as described in Fig. 5C. Cells were exposed to Ara-C or etoposide (VP16) for 1 h and evaluated 10 days later. Right panel: N-acetylcysteine treatment rescues the higher toxicity of Ara-C or etoposide on "Ubc9 DA" cells. Cells were treated with 0.5 mM NAC 16 h prior to addition of etoposide or Ara-C. Graphs represent means of three to four independent experiments, error bars represent S.E.M. * p < 0.05.


B Following treatment with 10 mM metformin overnight , NIH3T3 reporter were heat - shocked at 43 ° C for 30 min recovered at 37 ° C for 5 h . GFP induction was quantitated by flow cytometry . HS : heat shock .

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B Following treatment with 10 mM metformin overnight, NIH3T3 reporter cells were heat-shocked at 43°C for 30 min and recovered at 37°C for 5 h. GFP induction was quantitated by flow cytometry. HS: heat shock.


C in reporter treated with without 10 mM metformin overnight were detected by immunoblotting .

C Individual proteins in reporter cells treated with and without 10 mM metformin overnight were detected by immunoblotting.


D , E Following heat shock recovery for 4 h , mRNA in reporter treated with without 10 mM metformin overnight were quantitated by qRT - PCR ( mean ± SD , n = 3 , Student ' s t - , * * P < 0 . 01 , * * * P < 0 . 001 ) . β - actin was used as internal control .

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D, E Following heat shock and recovery for 4 h, mRNA levels in reporter cells treated with and without 10 mM metformin overnight were quantitated by qRT-PCR (mean ± SD, n = 3, Student's t-test, **P < 0.01, ***P < 0.001). β-actin was used as the internal control.


F Reporter were treated with 10 mM metformin overnight , viability was measured using Guava ViaCount ® reagents ( mean ± SD , n = 5 , n . s . , not significant , Student ' s t - ) .

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F Reporter cells were treated with 10 mM metformin overnight, and viability was measured using Guava ViaCount® reagents (mean ± SD, n = 5, n.s., not significant, Student's t-test).


G Left panel , graphic depiction of ELISA - binding . HSE : heat shock element . Right panel : Immediately following heat shock at 43 ° C for 30 min , of reporter treated with without 10 mM metformin were extracted for ( mean ± SD , n = 3 , Student ' s t - , * * P < 0 . 01 , * * * P < 0 . 001 ) . Experimental details are described in Materials Methods .

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G Left panel, graphic depiction of the ELISA-based DNA binding assay. HSE: heat shock element. Right panel: Immediately following heat shock at 43°C for 30 min, nuclear proteins of the reporter cells treated with and without 10 mM metformin were extracted for assay (mean ± SD, n = 3, Student's t-test, **P < 0.01, ***P < 0.001). Experimental details are described in Materials and Methods.


I HEK293T stably expressing a scramble or HSF1 - targeting shRNA were treated with 10 mM metformin overnight heat - shocked at 43 ° C for 30 min . were fixed for PLA to detect HSF1 - interactions . Experimental details are described in Materials Methods . Scale bars : 50 μm for low magnification 10 μm for high magnification .

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I HEK293T cells stably expressing a scramble or HSF1-targeting shRNA were treated with 10 mM metformin overnight and heat-shocked at 43°C for 30 min. Cells were fixed for PLA to detect HSF1-DNA interactions. Experimental details are described in Materials and Methods. Scale bars: 50 μm for low magnification and 10 μm for high magnification.


J Immediately following heat shock , of reporter treated with without 10 mM metformin were extracted for immunoblotting . LDH Lamin A / C were used as markers , respectively . C : cytoplasm ; N : nucleus .

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J Immediately following heat shock, cytoplasmic and nuclear proteins of the reporter cells treated with and without 10 mM metformin were extracted for immunoblotting. LDH and Lamin A/C were used as cytoplasmic and nuclear markers, respectively. C: cytoplasm; N: nucleus.


K Reporter mice ( ventral side shown ) were pre - treated with metformin for 3 days , 2 mg / mouse / day , via i . p . injection . Following a single i . p . injection of velcade ( 5 mg / kg ) recovery for 6 h , whole - luciferase activities were quantitated by bioluminescence imaging . photon flux of each mouse was expressed as photon / s / cm2 / steradian ( right panel , mean ± SD , n = 3 , one - way ANOVA , * P < 0 . 05 ; * * P < 0 . 01 ) .

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K Reporter mice (ventral side shown) were pre-treated with metformin for 3 days, 2 mg/mouse/day, via i.p. injection. Following a single i.p. injection of velcade (5 mg/kg) and recovery for 6 h, whole-body luciferase activities were quantitated by bioluminescence imaging. The photon flux of each mouse was expressed as photon/s/cm2/steradian (right panel, mean ± SD, n = 3, one-way ANOVA, *P < 0.05; **P < 0.01).


A - C Reporter were treated with without 1 μM A - 769662 for 3 h followed by heat shock at 43 ° C for 30 min recovery at 37 ° C for 5 h . Hspa1a Hspb1 mRNA were quantitated by qRT - PCR ( mean ± SD , n = 3 , Student ' s t - , * * * P < 0 . 001 ) . ACC phosphorylation was detected by immunoblotting ( C ) .

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A-C Reporter cells were treated with and without 1 μM A-769662 for 3 h followed by heat shock at 43°C for 30 min and recovery at 37°C for 5 h. Hspa1a and Hspb1 mRNA levels were quantitated by qRT-PCR (mean ± SD, n = 3, Student's t-test, ***P < 0.001). ACC phosphorylation was detected by immunoblotting (C).


D - F MEFs were derived from Ampkα1fl / fl ; Ampkα2fl / fl transduced with either adenoviral GFP or Cre to delete Ampkα1 / 2 in vitro . AMPKα were detected 4 days after transductions ( D )

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D-F Primary MEFs were derived from Ampkα1fl/fl; Ampkα2fl/fl embryos and transduced with either adenoviral GFP or Cre to delete Ampkα1/2 in vitro. AMPKα levels were detected 4 days after transductions (D)


Following viral transductions , Hsp mRNA were quantitated by qRT - PCR in MEFs with without 10 mM metformin treatment overnight followed by 43 ° C heat shock for 30 min recovery for 4 h ( mean ± SD , n = 3 , one - way ANOVA , n . s . , not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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Following viral transductions, Hsp mRNA levels were quantitated by qRT-PCR in MEFs with and without 10 mM metformin treatment overnight followed by 43°C heat shock for 30 min and recovery for 4 h (mean ± SD, n = 3, one-way ANOVA, n.s., not significant, *P < 0.05, **P < 0.01, ***P < 0.001).


G Transcriptional activities of HSF1 were measured by a dual reporter system consisting of two plasmids : HSF1 - dependent reporter pHSE - SEAP , in which ideal HSEs drive expression of secreted alkaline phosphatase ( SEAP ) , control reporter pCMV - Gaussia Luc , in which a CMV promoter drives expression of secreted Gaussia luciferase . In HEK293T , either LacZ or GST - AMPKα1CA plasmid was co - transfected with two reporter plasmids . After 24 h , transfected were heat - shocked at 44 ° C for 45 min . culture supernatants were collected to measure SEAP luciferase activities 24 h after heat shock . SEAP signals were normalized to Gaussia luciferase signals ( mean ± SD , n = 5 , Student ' s t - , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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G Transcriptional activities of HSF1 were measured by a dual reporter system consisting of two plasmids: the HSF1-dependent reporter pHSE-SEAP, in which ideal HSEs drive the expression of secreted alkaline phosphatase (SEAP), and the control reporter pCMV-Gaussia Luc, in which a CMV promoter drives the expression of secreted Gaussia luciferase. In HEK293T cells, either LacZ or GST-AMPKα1CA plasmid was co-transfected with the two reporter plasmids. After 24 h, the transfected cells were heat-shocked at 44°C for 45 min. The culture supernatants were collected to measure SEAP and luciferase activities 24 h after heat shock. SEAP signals were normalized to Gaussia luciferase signals (mean ± SD, n = 5, Student's t-test, **P < 0.01, ***P < 0.001).


H , I Following treatment with 10 mM metformin overnight , endogenous AMPKα HSF1 were co - precipitated using anti - AMPKα agarose conjugates from lysates of immortalized Hsf1 + / + MEFs ( H ) .

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H, I Following treatment with 10 mM metformin overnight, endogenous AMPKα and HSF1 proteins were co-precipitated using anti-AMPKα agarose conjugates from lysates of immortalized Hsf1+/+MEFs (H).


HC : heavy chain . Endogenous HSF1 - AMPKα interactions in immortalized Hsf1 + / + Hsf1 − / − MEFs were visualized in situ by PLA ( I ) . Experimental details are described in Materials Methods . Scale bars : 50 μm .

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HC: heavy chain. Endogenous HSF1-AMPKα interactions in immortalized Hsf1+/+ and Hsf1−/− MEFs were visualized in situ by PLA (I). Experimental details are described in Materials and Methods. Scale bars: 50 μm.


A In HEK293T that stably express an shRNA targeting 3 ′ UTR of human HSF1 , HSF1WT or HSF1S121A was expressed . Following treatment with 10 mM metformin overnight , of HSF1 phosphorylation at Ser121 were detected by immunoblotting using a specific phospho - HSF1 Ser121 antibody ( A8041 , Biotechnology ) .

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A In HEK293T cells that stably express an shRNA targeting the 3′ UTR of human HSF1, HSF1WT or HSF1S121A was expressed. Following treatment with 10 mM metformin overnight, the levels of HSF1 phosphorylation at Ser121 were detected by immunoblotting using a specific phospho-HSF1 Ser121 antibody (A8041, Assay Biotechnology).


B Ampkα1fl / fl ; Ampkα2fl / fl MEFs were transduced with adenoviral GFP or Cre . Following treatment with 10 mM metformin overnight or treatment with 10 μM A - 769662 for 3 h , HSF1 Ser121 phosphorylation was examined by immunoblotting .

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B Primary Ampkα1fl/fl; Ampkα2fl/fl MEFs were transduced with adenoviral GFP or Cre. Following treatment with 10 mM metformin overnight or treatment with 10 μM A-769662 for 3 h, HSF1 Ser121 phosphorylation was examined by immunoblotting.


C HSF1WT or HSF1S121A was co - expressed with either GFP or GST - AMPKαCA in HSF1 - deficient HEK293T . HSF1 Ser121 phosphorylation was examined by immunoblotting .

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C HSF1WT or HSF1S121A was co-expressed with either GFP or GST-AMPKαCA in HSF1-deficient HEK293T cells. HSF1 Ser121 phosphorylation was examined by immunoblotting.


D AMPK complexes were immunoprecipitated from HEK293T treated with without 10 mM metformin overnight . Aliquots of AMPK complexes were incubated with 400 ng purified recombinant His - tagged HSF1 with without 100 μM AMP or 20 μM compound C ( CC ) . HSF1 Ser121 phosphorylation was detected by immunoblotting .

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D AMPK complexes were immunoprecipitated from HEK293T cells treated with and without 10 mM metformin overnight. Aliquots of AMPK complexes were incubated with 400 ng purified recombinant His-tagged HSF1 proteins with and without 100 μM AMP or 20 μM compound C (CC). HSF1 Ser121 phosphorylation was detected by immunoblotting.


E , F HSF1 activities were measured by dual reporter system in HSF1 - deficient HEK293T . Either FLAG - HSF1WT or FLAG - HSF1S121A was co - expressed with LacZ orAMPKα1CA ( E ) .

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E, F HSF1 activities were measured by the dual reporter system in HSF1-deficient HEK293T cells. Either FLAG-HSF1WT or FLAG-HSF1S121A was co-expressed with LacZ orAMPKα1CA (E).


E , F HSF1 activities were measured by dual reporter system in HSF1 - deficient HEK293T . Either FLAG - HSF1WT or FLAG - HSF1S121A was co - expressed with LacZ or AMPKα1CA ( E ) . Following expression of HSF1WT or HSF1S121A , were treated with without 10 mM metformin overnight ( F ) ( mean ± SD , n = 5 , one - way ANOVA , n . s . , not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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E, F HSF1 activities were measured by the dual reporter system in HSF1-deficient HEK293T cells. Either FLAG-HSF1WT or FLAG-HSF1S121A was co-expressed with LacZ or AMPKα1CA (E). Following expression of HSF1WT or HSF1S121A, cells were treated with and without 10 mM metformin overnight (F) (mean ± SD, n = 5, one-way ANOVA, n.s., not significant, *P < 0.05, **P < 0.01, ***P < 0.001).


G , H FLAG - HSF1WT or FLAG - HSF1S121A plasmids were co - transfected with LacZ or AMPKα1CA plasmids into HSF1 - deficient HEK293T for 3 days ( G ) . Following transfection with FLAG - HSF1WT or FLAG - HSF1S121A plasmids for 3 days , HSF1 - deficient HEK293T were treated with without 10 mM metformin overnight ( H ) . were extracted to measure HSF1 - binding by ELISA - binding using anti - FLAG antibodies ( mean ± SD , n = 3 , one - way ANOVA , n . s . , not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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G, H FLAG-HSF1WT or FLAG-HSF1S121A plasmids were co-transfected with LacZ or AMPKα1CA plasmids into HSF1-deficient HEK293T cells for 3 days (G). Following transfection with FLAG-HSF1WT or FLAG-HSF1S121A plasmids for 3 days, HSF1-deficient HEK293T cells were treated with and without 10 mM metformin overnight (H). Nuclear proteins were extracted to measure HSF1-DNA binding by the ELISA-based DNA binding assay using anti-FLAG antibodies (mean ± SD, n = 3, one-way ANOVA, n.s., not significant, *P < 0.05, **P < 0.01, ***P < 0.001).


I , J Following transfections metformin treatment as described in ( G ) ( H ) , were extracted for immunoblotting using anti - FLAG antibodies .

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I, J Following transfections and metformin treatment as described in (G) and (H), cytoplasmic and nuclear proteins were extracted for immunoblotting using anti-FLAG antibodies.


A , B NIH3T3 reporter were cultured overnight in DMEM with without leucine ( A ) or 4 . 5 g / l glucose ( B ) . Following heat shock at 43 ° C for 30 min recovery for 5 h , GFP induction was quantitated by flow cytometry .

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A, B NIH3T3 reporter cells were cultured overnight in DMEM with and without leucine (A) or 4.5 g/l glucose (B). Following heat shock at 43°C for 30 min and recovery for 5 h, GFP induction was quantitated by flow cytometry.


C , D were extracted from reporter with without nutrient deprivations overnight for ELISA - binding ( mean ± SD , n = 3 , Student ' s t - , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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C, D Nuclear proteins were extracted from reporter cells with and without nutrient deprivations overnight for the ELISA-based DNA binding assay (mean ± SD, n = 3, Student's t-test, *P < 0.05, **P < 0.01, ***P < 0.001).


E , F Following nutrient deprivations overnight , reporter were heat - shocked at 43 ° C for 30 min recovered overnight . were detected by immunoblotting .

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E, F Following nutrient deprivations overnight, the reporter cells were heat-shocked at 43°C for 30 min and recovered overnight. Individual proteins were detected by immunoblotting.


G , H Reporter were subjected to nutrient starvation for indicated time . AMPKα Thr172 HSF1 Ser121 phosphorylation was measured by immunoblotting .

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G, H Reporter cells were subjected to nutrient starvation for the indicated time. AMPKα Thr172 and HSF1 Ser121 phosphorylation was measured by immunoblotting.


I , J HEK293T were sequentially transfected with control or AMPKα1 / 2 - targeting siRNAs dual HSF1 reporter plasmids . 24 h later , transfected were starved overnight before reporter activities were measured ( mean ± SD , n = 6 , Student ' s t - , n . s . , not significant , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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I, J HEK293T cells were sequentially transfected with control or AMPKα1/2-targeting siRNAs and dual HSF1 reporter plasmids. 24 h later, transfected cells were starved overnight before reporter activities were measured (mean ± SD, n = 6, Student's t-test, n.s., not significant, **P < 0.01, ***P < 0.001).


K , L FLAG - HSF1WT or FLAG - HSF1S121A plasmids were co - transfected with dual HSF1 reporter plasmids into HSF1 - deficient HEK293T for 2 days , followed by overnight starvation ( mean ± SD , n = 5 , Student ' s t - , n . s . , not significant , * P < 0 . 05 , * * P < 0 . 01 ) .

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K, L FLAG-HSF1WT or FLAG-HSF1S121A plasmids were co-transfected with the dual HSF1 reporter plasmids into HSF1-deficient HEK293T cells for 2 days, followed by overnight starvation (mean ± SD, n = 5, Student's t-test, n.s., not significant, *P < 0.05, **P < 0.01).


A Reporter were heat - shocked at 43 ° C for indicated time . AMPKα Thr172 HSF1 Ser121 phosphorylation was measured by immunoblotting .

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A Reporter cells were heat-shocked at 43°C for the indicated time. AMPKα Thr172 and HSF1 Ser121 phosphorylation was measured by immunoblotting.


B Immediately following heat shock at 43 ° C for 40 min , AMPK complexes were immunoprecipitated from HEK293 in vitro kinase assays were performed as described in Figure 3D using recombinant human ACC1 as substrates .

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B Immediately following heat shock at 43°C for 40 min, AMPK complexes were immunoprecipitated from HEK293 cells and in vitro kinase assays were performed as described in Figure 3D using recombinant human ACC1 proteins as substrates.


C Following heat shock at 43 ° C for 40 min , both detergent - soluble detergent - insoluble fractions of were extracted from HEK293 as described in Materials Methods for immunoblotting .

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C Following heat shock at 43°C for 40 min, both detergent-soluble and detergent-insoluble fractions of cellular proteins were extracted from HEK293 cells as described in Materials and Methods for immunoblotting.


D , E Immediately following heat shock at 43 ° C for 40 min , HEK293 were subjected to nutrient deprivations for 4 h . AMPKα Thr172 ACC Ser79 phosphorylation was measured by immunoblotting .

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D, E Immediately following heat shock at 43°C for 40 min, HEK293 cells were subjected to nutrient deprivations for 4 h. AMPKα Thr172 and ACC Ser79 phosphorylation was measured by immunoblotting.


F - H HSF1WT or HSF1S121A plasmids were transfected into HSF1 - deficient HEK293T . Transfected were heat - shocked at 43 ° C for 40 min . HSP mRNAs were quantitated by qRT - PCR following overnight recovery . HSF1 - binding was measured immediately after heat shock ( mean ± SD , n = 3 , Student ' s t - , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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F-H HSF1WT or HSF1S121A plasmids were transfected into HSF1-deficient HEK293T cells. Transfected cells were heat-shocked at 43°C for 40 min. HSP mRNAs were quantitated by qRT-PCR following overnight recovery. HSF1-DNA binding was measured immediately after heat shock (mean ± SD, n = 3, Student's t-test, **P < 0.01, ***P < 0.001).


A , B Following treatment with 10 μM metformin for 3 days , of HSF1 binding to endogenous HSP promoters were quantitated by IP ( mean ± SD , n = 3 , one - way ANOVA , * * P < 0 . 01 , * * * P < 0 . 001 ) . Normal rabbit IgG served as negative control ( A ) . Following same treatment , HSP mRNA were quantitated by qRT - PCR ( mean ± SD , n = 3 , Student ' s t - , * * * P < 0 . 001 ) ( B ) .

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A, B Following treatment with 10 μM metformin for 3 days, the levels of HSF1 binding to endogenous HSP promoters were quantitated by chromatin IP (mean ± SD, n = 3, one-way ANOVA, **P < 0.01, ***P < 0.001). Normal rabbit IgG served as the negative control (A). Following the same treatment, HSP mRNA levels were quantitated by qRT-PCR (mean ± SD, n = 3, Student's t-test, ***P < 0.001) (B).


C , D Following transfection with AMPKα1 / 2 - targeting siRNAs for 2 days , WM115 were treated with 10 μM metformin for 3 days . HSP mRNA were quantitated by qRT - PCR ( mean ± SD , n = 6 , Student ' s t - , n . s . , not significant , * P < 0 . 05 , * * P < 0 . 01 ) .

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C, D Following transfection with AMPKα1/2-targeting siRNAs for 2 days, WM115 cells were treated with 10 μM metformin for 3 days. HSP mRNA levels were quantitated by qRT-PCR (mean ± SD, n = 6, Student's t-test, n.s., not significant, *P < 0.05, **P < 0.01).


E Following treatment with 10 μM metformin for 7 days , in WM115 S462 were measured by immunoblotting .

E Following treatment with 10 μM metformin for 7 days, protein levels in WM115 and S462 cells were measured by immunoblotting.


F , G Following treatment with 10 μM metformin for 7 days , of polyubiquitinated were detected in both detergent - soluble detergent - insoluble fractions in WM115 A2058 using a Lys48 - specific polyubiquitin antibody .

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F, G Following treatment with 10 μM metformin for 7 days, levels of polyubiquitinated proteins were detected in both detergent-soluble and detergent-insoluble fractions in WM115 and A2058 cells using a Lys48-specific polyubiquitin antibody.


H A2058 were treated with 10 μM metformin alone or co - treated with 2 μM compound C for 7 days . were detected by immunoblotting .

H A2058 cells were treated with 10 μM metformin alone or co-treated with 2 μM compound C for 7 days. Individual proteins were detected by immunoblotting.


I HEK293T stably expressing either scramble or HSF1 - targeting shRNAs were treated with 10 μM metformin for 7 days . were detected by immunoblotting .

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I HEK293T cells stably expressing either scramble or HSF1-targeting shRNAs were treated with 10 μM metformin for 7 days. Individual proteins were detected by immunoblotting.


J In HEK293T , a plasmid encoding HA - polyQ79 - GFP was co - transfected with indicated plasmids . Following treatment with without 10 μM metformin for 5 days , sizes of in detergent - insoluble fractions were quantitated by a Multisizer ™ 3 Coulter Counter . Experimental details are described in Materials Methods .

J In HEK293T cells, a plasmid encoding HA-polyQ79-GFP was co-transfected with the indicated plasmids. Following treatment with and without 10 μM metformin for 5 days, the sizes of aggregates in detergent-insoluble fractions were quantitated by a Multisizer™ 3 Coulter Counter. Experimental details are described in Materials and Methods.


K , L Immortalized RAS - transformed MEFs were treated with either 10 μM metformin ( K ) or different concentrations of glucose ( L ) for 7 days . were detected by immunoblotting .

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K, L Immortalized and RAS-transformed MEFs were treated with either 10 μM metformin (K) or different concentrations of glucose (L) for 7 days. Individual proteins were detected by immunoblotting.


M Following transfection with LacZ or polyQ79 , HEK293T were grown under different concentrations of glucose for 5 days before measuring aggregate .

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M Following transfection with LacZ or polyQ79, HEK293T cells were grown under different concentrations of glucose for 5 days before measuring aggregate size.


A , B A2058 were transduced with lentiviral scramble or HSF1 - targeting ( hA6 hA9 ) shRNAs . ( A ) numbers were quantitated by Hoechst 33342 ( mean ± SD , n = 4 , two - way ANOVA , * * * P < 0 . 001 ) . ( B ) were measured by immunoblotting .

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A, B A2058 cells were transduced with lentiviral scramble or HSF1-targeting (hA6 and hA9) shRNAs. (A) Cell numbers were quantitated by Hoechst 33342 DNA staining (mean ± SD, n = 4, two-way ANOVA, ***P < 0.001). (B) Protein levels were measured by immunoblotting.


C A2058 stably expressing LacZ or HSF1 were grown in medium containing 4 . 5 , 1 . 0 , or 0 . 45 g / l glucose . Following treatment with 10 μM metformin , proliferation was measured by Hoechst 33342 ( mean ± SD , n = 5 , two - way ANOVA , * * * P < 0 . 001 ) .

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C A2058 cells stably expressing LacZ or HSF1 were grown in medium containing 4.5, 1.0, or 0.45 g/l glucose. Following treatment with 10 μM metformin, cell proliferation was measured by Hoechst 33342 DNA staining (mean ± SD, n = 5, two-way ANOVA, ***P < 0.001).


D , E 1 × 106 LacZ - or HSF1 - expressing A2058 were transplanted into NOD / SCID mice . One day after transplantation , metformin was administered via drinking water at 1 mg / ml . incidence ( log - rank ; D ) volumes ( E ) were measured ( mean ± , two - way ANOVA n . s . , not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . growth curves were fitted to exponential growth models to calculate doubling time ( DT ) .

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D, E 1 × 106 LacZ- or HSF1-expressing A2058 cells were transplanted into female NOD/SCID mice. One day after transplantation, metformin was administered via drinking water at 1 mg/ml. Tumor incidence (log-rank test; D) and volumes (E) were measured (mean ± SEM, two-way ANOVA n.s., not significant, *P < 0.05, **P < 0.01, ***P < 0.001). Tumor growth curves were fitted to exponential growth models to calculate tumor doubling time (DT).


F were measured by immunoblotting using lysates of three tumors from each treatment group .

F Individual proteins were measured by immunoblotting using lysates of three tumors from each treatment group.


G were measured by immunoblotting using lysates of mouse liver tissues .

G Protein levels were measured by immunoblotting using lysates of mouse liver tissues.


H A2058 stably expressing FLAG - HSF1WT or FLAG - HSF1S121A were treated with 10 μM metformin for 7 days . were measured by immunoblotting .

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H A2058 cells stably expressing FLAG-HSF1WT or FLAG-HSF1S121A were treated with 10 μM metformin for 7 days. Protein levels were measured by immunoblotting.


I A2058 stably expressing HSF1WT or HSF1S121A were grown in Corning ® ultra - low attachment 96 - well culture plates with 10 μM metformin different concentrations of glucose for 7 days , 10 , 000 per well . Viable were counted by Guava flow cytometer using ViaCount ® reagent ( mean ± , n = 8 , Student ' s t - , n . s . , not significant , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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I A2058 cells stably expressing HSF1WT or HSF1S121A were grown in Corning®ultra-low attachment 96-well culture plates with 10 μM metformin and different concentrations of glucose for 7 days, 10,000 cells per well. Viable cells were counted by Guava flow cytometer using ViaCount® reagent (mean ± SEM, n = 8, Student's t-test, n.s., not significant, **P < 0.01, ***P < 0.001).


J Tukey box plots showing inverse correlation between AMPKα Thr172 phosphorylation HSP mRNA expression in ccRCC . were stratified on median value of AMPKα Thr172 phosphorylation reverse - phase array ( RPPA ) scores ( Student ' s t - ) .

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J Tukey box plots showing the inverse correlation between AMPKα Thr172 phosphorylation and HSP mRNA expression in ccRCC. Patients were stratified on the median value of AMPKα Thr172 phosphorylation reverse-phase protein array (RPPA) scores (Student's t-test).


( A ) Localization of endogenous SHP - 1 in hematopoietic non - hematopoietic was determined by confocal imaging after with SHP - 1 antibody . DAPI was used for , Scale bar : 5µm . intensities along drawn in a representative were obtained using Zen software co - localization plots were made using GraphPad ( Red : SHP - 1 , Blue : DAPI ) .

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(A) Localization of endogenous SHP-1 in hematopoietic and non-hematopoietic cells was determined by confocal imaging after staining with SHP-1 antibody. DAPI was used for nuclear staining, Scale bar:5µm. Fluorescence intensities along the line drawn in a representative cell were obtained using Zen software and the co-localization plots were made using GraphPad (Red: SHP-1, Blue: DAPI).


( C ) SFB tagged PAF1 , SFB - LEO1 , SFB - CTR9 , SFB - CDC73 , SFB - RTF1 , SFB - WDR61 along with Myc SHP - 1 or Myc PTPN7 were transfected in 293T interaction of SHP - 1 with PAF components was detected by immunoblotting with Myc antibody after S beads pulldown in presence of TurboNuclease .

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(C) SFB tagged PAF1, SFB-LEO1, SFB-CTR9, SFB-CDC73, SFB-RTF1, SFB-WDR61 along with Myc SHP-1 or Myc PTPN7 were transfected in 293T cells and interaction of SHP-1 with PAF complex components was detected by immunoblotting with Myc antibody after S protein beads pulldown in presence of TurboNuclease.


( D ) SFB empty vector , SFB SHP - 1 or SFB PTPN7 was co - transfected with Myc tagged H2B in 293T . interaction of H2B with SHP - 1 was detected through immunoblotting using Myc antibody after pull down with streptavidin sepharose beads .

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(D) SFB empty vector, SFB SHP-1 or SFB PTPN7 was co-transfected with Myc tagged H2B in 293T cells. The interaction of H2B with SHP-1 was detected through immunoblotting using Myc antibody after pull down with streptavidin sepharose beads.


( E ) Endogenous association of SHP - 1 with Paf1 subunits ( CDC73 , RTF1 , WDR61 , LEO1 , Paf1 , CTR9 ) was detected by immunoblotting after performing immunoprecipitation with either IgG or SHP - 1 antibody .

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(E) Endogenous association of SHP-1 with Paf1 complex subunits (CDC73, RTF1, WDR61, LEO1, Paf1, CTR9) was detected by immunoblotting after performing immunoprecipitation with either IgG or SHP-1 antibody.


( F ) Immunoprecipitation ( IP ) with control IgG or anti - SHP - 1 antibody was performed with extracts prepared from MCF7 endogenous association of H2B with SHP - 1 was detected by immunoblotting .

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(F) Immunoprecipitation (IP) with control IgG or anti-SHP-1 antibody was performed with extracts prepared from MCF7 cells and endogenous association of H2B with SHP-1 was detected by immunoblotting.


( G ) Glutathione Sepharose beads immobilized with bacterially expressed recombinant GST or GST - H2B were incubated with bacterially purified recombinant MBP - SHP - 1 . association of SHP - 1 with H2B was detected by immunoblotting with MBP antibody . Expression of GST , GST - H2B MBP SHP - 1 was shown by Coomassie .

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(G) Glutathione Sepharose beads immobilized with bacterially expressed recombinant GST or GST-H2B proteins were incubated with bacterially purified recombinant MBP-SHP-1. The association of SHP-1 with H2B was detected by immunoblotting with MBP antibody. Expression of GST, GST-H2B and MBP SHP-1 was shown by Coomassie staining.


( A ) extracts of WT SHP - 1 knockout ( derived from two independent guide RNAs ) were made of H2BUb were detected .

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(A) Nuclear extracts of WT and SHP-1 knockout cells (derived from two independent guide RNAs) were made and the levels of H2BUb were detected.


( B ) Wild type ( WT ) 293T along with SHP - 1 KO transfected with either Myc vector , Myc SHP - 1 ∆ SH2 or Myc SHP - 1 NLS mutant were lysed . H2BUb were determined using specific antibody .

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(B) Wild type (WT) 293T cells along with SHP-1 KO cells transfected with either Myc vector, Myc SHP-1 ∆SH2 or Myc SHP-1 NLS mutant were lysed. H2BUb levels were determined using specific antibody.


( C ) WT SHP - 1 KO 293T transfected with Myc vector , Myc SHP - 1 or Myc SHP - 1 C / S ( C453S ) mutant were lysed . H2BUb were determined using specific antibody .

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(C) WT and SHP-1 KO 293T cells transfected with Myc vector, Myc SHP-1 or Myc SHP-1 C/S (C453S) mutant were lysed. H2BUb levels were determined using specific antibody.


( D ) H2B WT along with indicated Y / F Y / D mutants were transfected into 293T . Histone extract was prepared from transfected with each construct subjected to western blotting with anti - H2B anti - H2BUb antibodies .

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 B-MEASURED_VAR O O B-MEASURED_VAR O O

(D) H2B WT along with the indicated Y/F and Y/D mutants were transfected into 293T cells. Histone extract was prepared from cells transfected with each construct and subjected to western blotting with anti-H2B and anti-H2BUb antibodies.