( e ) Immunostaining with pax6 in E16 electroporated embryo brains . White arrows represent PAX6 , GFP double - positive cells . 1 , 2 are 2x enlarged as the dashed boxes showed . Scale bar : 50 μm . ( f ) Statistical analysis of the percentage of PAX6 + GFP + cells among the total GFP + cells . n = 3 brains for each group . 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 ( * * ) , 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunostaining with pax6 in E16 electroporated embryo brains. White arrows represent PAX6, GFP double-positive cells. 1,2 are 2x enlarged as the dashed boxes showed. Scale bar: 50 μm. (f) Statistical analysis of the percentage of PAX6+GFP+ cells among the total GFP+ cells. n=3 brains for each group. 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(**), p<0.001(***).


( g ) Immunostaining with sox2 and pax6 in E16 electroporated embryo brains . White arrows represent GFP , sox2 double positive cells ; Red arrows represent GFP , TBR2 double positive cells . area . Scale bar : 50 μm . ( h ) Statistical analysis of the percentage of SOX2 + GFP + cells among the total GFP + cells . n = 8 brains for each group . ( i ) Statistical analysis of the percentage of TBR2 + GFP + cells among the total GFP + cells . n = 5 brains for each group . 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 ( * * ) , 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunostaining with sox2 and pax6 in E16 electroporated embryo brains. White arrows represent GFP, sox2 double positive cells; Red arrows represent GFP, TBR2 double positive cells. area. Scale bar: 50 μm. (h) Statistical analysis of the percentage of SOX2+GFP+ cells among the total GFP+ cells. n=8 brains for each group. (i) Statistical analysis of the percentage of TBR2+GFP+ cells among the total GFP+ cells. n=5 brains for each group. 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(**), p<0.001(***).


( a ) SATB2 staining revealed a reduction in SATB2 and GFP colocalization when TCF20 was depleted . White arrows represent SATB2 , GFP double - positive cells . ( b ) Statistical analysis of the SATB2 GFP double - positive cell ratio in GFP positive cells . n = 3 brains . 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 ( * * ) , p < 0 . 001 ( * * * ) . Scale bar : 25

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) SATB2 staining revealed a reduction in SATB2 and GFP colocalization when TCF20 was depleted. White arrows represent SATB2, GFP double-positive cells. (b) Statistical analysis of the SATB2 GFP double-positive cell ratio in GFP positive cells. n=3 brains. 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(**), p<0.001(***). Scale bar: 25 μm


( c ) Staining with CTIP2 and TBR1 antibodies shows TCF20 knockdown decreases the colocalization of CTIP2 and TBR1 with GFP in E13 . 5 - E16 . 5 brains . White arrows represent CTIP2 , GFP double - positive cells . White arrow heads represent TBR1 , GFP double - positive cells . ( d , e ) Statistical analysis of CTIP2 , GFP and TBR1 , GFP double - positive cell ratio in GFP positive cells . n = 3 brains . 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 ( * * ) , p < 0 . 001 ( * * * ) . Scale bar : ( d ) 50

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Staining with CTIP2 and TBR1 antibodies shows TCF20 knockdown decreases the colocalization of CTIP2 and TBR1 with GFP in E13.5-E16.5 brains. White arrows represent CTIP2, GFP double-positive cells. White arrow heads represent TBR1, GFP double-positive cells. (d, e) Statistical analysis of CTIP2, GFP and TBR1, GFP double-positive cell ratio in GFP positive cells. n=3 brains. 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(**), p<0.001(***). Scale bar: (d) 50 μm


( f ) A diagram of the BrdU labeling time point in electroporation experiments . ( g ) Immunostaining of BrdU . White arrows represent BrdU GFP double - positive cells . ( h ) Percentage of BrdU and GFP double - positive cells in GFP - positive cells n = 3 brains for each group 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 ( * * ) , p < 0 . 001 ( * * * ) . Scale bar : f ) 25

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

(f) A diagram of the BrdU labeling time point in electroporation experiments. (g) Immunostaining of BrdU. White arrows represent BrdU GFP double-positive cells. (h) Percentage of BrdU and GFP double-positive cells in GFP-positive cells n=3 brains for each group 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(**), p<0.001(***). Scale bar: f) 25 μm


( b ) Western blotting analysis of TCF20 in TCF20 WT , HET , KO mice reveals that TCF20 was deleted . ( c ) Statistical analysis of the normalized band intensity of TCF20 . n = 3 samples for each genotype . 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 ( * * ) , 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blotting analysis of TCF20 in TCF20 WT, HET, KO mice reveals that TCF20 was deleted. (c) Statistical analysis of the normalized band intensity of TCF20. n=3 samples for each genotype. 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(**), p<0.001(***).


( d ) RT - qPCR analysis of TCF20 in TCF20 WT , HET , and KO mice reveals that TCF20 was deleted . n = 4 samples . 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 ( * * ) , 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RT-qPCR analysis of TCF20 in TCF20 WT, HET, and KO mice reveals that TCF20 was deleted. n=4 samples. 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(**), p<0.001(***).


( e ) Immunostaining of E16 WT and KO mouse brain slices revealed that TCF20 was successfully knocked out . Scale bar : 50 μm . ( f ) Statistical analysis of the normalized fluorescence intensity of TCF20 . n = 6 samples for each genotype . 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 ( * * ) , 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunostaining of E16 WT and KO mouse brain slices revealed that TCF20 was successfully knocked out. Scale bar: 50 μm. (f) Statistical analysis of the normalized fluorescence intensity of TCF20. n=6 samples for each genotype. 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(**), p<0.001(***).


( g ) Western blotting analysis of neural proliferation and differentiation markers in WT HET and KO mice showed that TCF20 deficits promote cell proliferation and inhibit cell differentiation .

O O O O O O 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) Western blotting analysis of neural proliferation and differentiation markers in WT HET and KO mice showed that TCF20 deficits promote cell proliferation and inhibit cell differentiation.


( h ) Immunostaining of different cortical layer markers TBR1 , CTIP2 , and SATB2 , White dash line represents the positive area . Scale bar : 50 μm . ( i - k ) Statistical analysis of different cells layers in TCF20 WT , HET and KO mice . n = 7 brains of each group . 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 ( * * ) , 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunostaining of different cortical layer markers TBR1, CTIP2, and SATB2, White dash line represents the positive area. Scale bar: 50 μm. (i-k) Statistical analysis of different cells layers in TCF20 WT, HET and KO mice. n=7 brains of each group. 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(**), p<0.001(***).


( l ) TCF20 deletion impairs neurogenesis and results in abnormal cell distribution . Scale bar : 50 μm . ( m ) Statistical analysis of GFP + distribution in different zones . n = 3 brains for each genotype . 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 ( * * ) , 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(l) TCF20 deletion impairs neurogenesis and results in abnormal cell distribution. Scale bar: 50 μm. (m) Statistical analysis of GFP+ distribution in different zones. n=3 brains for each genotype. 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(**), p<0.001(***).


( a ) Diagram of pup ultrasonic vocalization at the frequency level after Fourier transforms ( b - d ) ( b ) Total call durations , ( c ) Call durations and ( d ) Call numbers of WT , HET , and KO mouse pups . nWT = 13 mice , nHET = 16 mice , and nKO = 18 mice . 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 ( * * ) , 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Diagram of pup ultrasonic vocalization at the frequency level after Fourier transforms (b-d) (b) Total call durations, (c) Call durations and (d) Call numbers of WT, HET, and KO mouse pups. nWT=13 mice, nHET=16 mice, and nKO=18 mice. 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(**), p<0.001(***).


( e ) A sketch of the mouse trace in the open field test . ( f - h ) ( f ) Statistical analysis of time spent in the center area , ( g ) Statistical analysis of total mouse movement distance in 5 min , ( h ) The ratio of mice in the center area movement distance to the total distance . n for WT = 15 adult mice , n for HET = 12 adult mice . 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 ( * * ) , 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) A sketch of the mouse trace in the open field test. (f-h) (f)Statistical analysis of time spent in the center area, (g) Statistical analysis of total mouse movement distance in 5 min, (h) The ratio of mice in the center area movement distance to the total distance. n for WT=15 adult mice, n for HET=12 adult mice. 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(**), p<0.001(***).


( i ) Statistical analysis of the Y maze test . S , O , and N represent the start Arm , old Arm and new Arm . The percent of time that mice stayed in each arm was recorded and analyzed ; n for WT = 11 adult mice , n for HET = 11 adult mice . Whiskers represent the minimum to maximum value ; central lines represent median values ; boxes represent the values between upper quartile and lower quartile . ( j ) Results of the elevated - plus maze . Time that mice spent in open arms ; n for WT = 8 adult mice , and n for HET = 11 adult mice . ( k ) Results of elevated - plus maze . Time that mice spent in closed arms ; n for WT = 8 adult mice , and n for HET = 11 adult mice . 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 ( * * ) , 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Statistical analysis of the Y maze test. S, O, and N represent the start Arm, old Arm and new Arm. The percent of time that mice stayed in each arm was recorded and analyzed; n for WT=11 adult mice, n for HET=11 adult mice. Whiskers represent the minimum to maximum value; central lines represent median values; boxes represent the values between upper quartile and lower quartile. (j) Results of the elevated-plus maze. Time that mice spent in open arms; n for WT=8 adult mice, and n for HET=11 adult mice. (k) Results of elevated-plus maze. Time that mice spent in closed arms ; n for WT=8 adult mice, and n for HET=11 adult mice.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(**), p<0.001(***).


( l ) Numbers of marbles buried in the marble burying test ; n for WT = 10 adult mice , and n for HET = 10 adult mice . 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 ( * * ) , 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(l) Numbers of marbles buried in the marble burying test; n for WT=10 adult mice, and n for HET=10 adult mice. 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(**), p<0.001(***).


( m ) Diagram of the three - chamber social interaction test and mouse movement trace . HET mice did not show preference or stay longer in the stranger1 or stranger2 chambers than the WT mice did in the two test phases . ( n ) Statistical analysis of the percentage of time spent in each chamber with one empty chamber and another chamber containing stranger1 ; n for WT = 16 adult mice , n for HET = 13 adult mice . ( o ) Comparison between the time that WT and HET mice spent in stranger1 chamber . n for WT = 16 adult mice , n for HET = 13 adult mice . ( p ) Time spent in each chamber when one contains familiar mouse stranger1 , as well as new mouse stranger2 . n for WT = 16 adult mice , n for HET = 13 adult mice . ( q ) Comparison between the time of WT and HET mice staying in the stranger2 chamber . n for WT = 16 adult mice , n for HET = 13 adult mice . 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 ( * * ) , 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(m) Diagram of the three-chamber social interaction test and mouse movement trace. HET mice did not show preference or stay longer in the stranger1 or stranger2 chambers than the WT mice did in the two test phases. (n) Statistical analysis of the percentage of time spent in each chamber with one empty chamber and another chamber containing stranger1; n for WT=16 adult mice, n for HET=13 adult mice. (o) Comparison between the time that WT and HET mice spent in stranger1 chamber. n for WT=16 adult mice, n for HET=13 adult mice. (p)Time spent in each chamber when one contains familiar mouse stranger1, as well as new mouse stranger2. n for WT=16 adult mice, n for HET=13 adult mice. (q) Comparison between the time of WT and HET mice staying in the stranger2 chamber. n for WT=16 adult mice, n for HET=13 adult mice. 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(**), p<0.001(***).


( c ) Heat map of significantly different genes .

(c) Heat map of significantly different genes.


( d ) TDG is significantly decreased after TCF20 deletion .

(d) TDG is significantly decreased after TCF20 deletion.


( e ) Volcano map showing that TDG is significantly decreased .

(e) Volcano map showing that TDG is significantly decreased.


( 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 ( * * ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ( * * ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ( * * * ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 , 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 ( * * * ) .

O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR 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 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 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 ( * * * ) .

O O O O O O O O O O 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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 . 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 ( * * * ) .

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 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

(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 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 ( * * * ) .

(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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ;

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 '

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

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

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 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 ;

O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O 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 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 ) .

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

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 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 ) .

O O O O O O O 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 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

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 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

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

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 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 ) .

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

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 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

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

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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 .

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

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 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 ;

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

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 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

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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 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 .

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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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 , 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 ) .

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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 , 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 . )

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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 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-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

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 - 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 .

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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 and D100A are equally active in CFP - RanGAPtail SUMOylation . Assays were with 35 nM Aos1 / Uba2 and 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 and 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..


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 -

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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 and 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 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 .

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

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 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

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

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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 : 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 .

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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 . 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 .

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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 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 .

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

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


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 .

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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 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 ) .

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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 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 .

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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 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 .

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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 , 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 .

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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 ) 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 ) .

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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 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 ) .

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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 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 )

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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 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 ) .

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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 : 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 ) .

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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 and HSF1 proteins 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 + / + and Hsf1 − / − MEFs were visualized in situ by PLA ( I ) . Experimental details are described in Materials and 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 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 ) .

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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 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 .

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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 cells . 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 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 .

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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 the dual reporter system in HSF1 - deficient HEK293T cells . 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 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 ) .

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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 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 ) .

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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 and metformin treatment as described in ( G ) and ( H ) , cytoplasmic and nuclear proteins 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 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 .

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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.