( D ) Neurodegenerative rough - eye phenotype in adults is alleviated by knockdown of Drosophila homologs of ufd - 2 / UBE4B spr - 5 / LSD1 - CG9934 Su ( Var ) 3 - 3 , respectively , compared to control ( CTRL ) . Eye - specific expression of TDP - 43M337V , FUSR521C , interference ( RNAi ) was driven by GMR - Gal4 .

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(D) Neurodegenerative rough-eye phenotype in adults is alleviated by the knockdown of the Drosophila homologs of ufd-2/UBE4B and spr-5/LSD1-CG9934 and Su(Var)3-3, respectively, compared to the control (CTRL). Eye-specific expression of TDP-43M337V, FUSR521C, and RNA interference (RNAi) was driven by GMR-Gal4.


( E ) Drosophila eye pigment quantitation . Data represent means ± . numerical data used to make this figure can be found in S1 Data .

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(E) Drosophila eye pigment quantitation. Data represent means ± SEM. The numerical data used to make this figure can be found in S1 Data.


( A ) Western blots of lysates derived from mock ( CTRL ) , single UBE4B or LSD1 , or double UBE4B LSD1 knockdowns . Supernatant ( S ) pellet ( P ) fractions were probed with indicated antibodies . While LSD1 or UBE4B single - knockdown reduces SOD1G85R in both supernatant pellet fractions , combined knockdown produces strongest reduction in . ( B ) of SOD1G85R by western blotting ( A ) . n = 3 ( supernatant ) ; n = 8 ( pellet ) .

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(A) Western blots of cell lysates derived from mock (CTRL), single UBE4B or LSD1, or double UBE4B and LSD1 knockdowns. Supernatant (S) and pellet (P) fractions were probed with indicated antibodies. While the LSD1 or UBE4B single-knockdown reduces the SOD1G85R aggregates in both supernatant and pellet fractions, the combined knockdown produces the strongest reduction in the aggregates. (B) Quantification of SOD1G85R protein levels by western blotting (A). n = 3 (supernatant); n = 8 (pellet).


( C ) Western blots of a representative cycloheximide chase experiment to determine SOD1 half - lives in double UBE4B LSD1 knockdown versus controls . ( D ) of SOD1G85R clearance , as analyzed by western blotting in ( C ) . graph indicates relative band of SOD1G85R at each chase time point . n = 5 ; Overall p = 0 . 02 ( paired t , CTRL versus UBE4B LSD1 double knockdown ) . p = 0 . 03 ( 3 h ) , p = 0 . 003 ( 6 h ) , p = 0 . 06 ( 9 h ) , p = 0 . 004 ( 12 - 21 h ) .

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(C) Western blots of a representative cycloheximide chase experiment to determine SOD1 protein half-lives in the double UBE4B and LSD1 knockdown cells versus controls. (D) Quantification of SOD1G85R clearance, as analyzed by western blotting in (C). The graph indicates the relative band intensity of SOD1G85R at each chase time point. n = 5; Overall p = 0.02 (paired t test, CTRL versus UBE4B and LSD1 double knockdown). Individual p = 0.03 (3 h), p = 0.003 (6 h), p = 0.06 (9 h), and p = 0.004 (12-21 h).


( E ) half - life of SOD1G85R is reduced from 8 . 5 h to 5 h upon knockdown of UBE4B LSD1 . Data represent means ± . numerical data used to make this figure can be found in S1 Data .

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(E) The half-life of SOD1G85R is reduced from 8.5 h to 5 h upon knockdown of UBE4B and LSD1. Data represent means ± SEM. The numerical data used to make this figure can be found in S1 Data.


( B ) volcano scatter plot indicates fold changes in of gene transcripts affected differentially by UBE4B LSD1 double knockdown versus control shRNA . Gray spots represent 22 , 148 annotated transcripts . Red spots are predicted p53 - activated targets , blue spots are predicted p53 - inhibited targets . enrichment of red spots in up - regulated genes ( right upper quadrant , > 1 . 2 - fold change , p ≤ 0 . 04 ) blue spots in down - regulated genes ( left upper quadrant ) indicates that p53 - mediated transcription is activated by UBE4B LSD1 double knockdown .

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(B) The volcano scatter plot indicates fold changes in the levels of gene transcripts affected differentially by the UBE4B and LSD1 double knockdown versus control shRNA. Gray spots represent 22,148 annotated transcripts. Red spots are predicted p53-activated targets, and blue spots are predicted p53-inhibited targets. The enrichment of red spots in the up-regulated genes (right upper quadrant, >1.2-fold change, p ≤ 0.04) and blue spots in the down-regulated genes (left upper quadrant) indicates that the p53-mediated transcription is activated by the UBE4B and LSD1 double knockdown.


C ) RT - qPCR validation of expression of representative p53 target genes in UBE4B LSD1 double - knockdown samples . n = ( 2 to 6 ) , p ≤ 0 . 04 .

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C) RT-qPCR validation of the expression levels of representative p53 target genes in the UBE4B and LSD1 double-knockdown samples. n = (2 to 6), p ≤ 0.04.


( D ) p53 level is significantly increased by UBE4B LSD1 double knockdown . Left : representative western blots showing p53 , knockdown of UBE4B LSD1 , actin control in HEK293T . Right : of p53 normalized against actin ( n = 3 ) .

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(D) The p53 protein level is significantly increased by UBE4B and LSD1 double knockdown. Left: representative western blots showing p53 levels, the knockdown of UBE4B and LSD1, and the actin control in HEK293T cells. Right: quantification of the p53 protein levels normalized against actin (n = 3).


E ) p53 - mediated transcriptional activity is measured by a luciferase reporter under control of a p53 - response element promoter , which was transfected into HEK293T 72 - 96 h after initiation of UBE4B LSD1 single or double knockdowns ( n = 6 ) .

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E) The p53-mediated transcriptional activity is measured by a luciferase reporter under the control of a p53-response element promoter, which was transfected into HEK293T cells 72-96 h after the initiation of the UBE4B and LSD1 single or double knockdowns (n = 6).


( F ) Increased interaction of p53 with its activating partner 53BP1 in response to UBE4B LSD1 knockdown . Left : representative western blots of 53BP1 co - immunoprecipitation . Right : of 53BP1 co - immunoprecipitation ( n = 2 ) . Data represent means ± .

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(F) Increased interaction of p53 with its activating partner 53BP1 in response to UBE4B and LSD1 knockdown. Left: representative western blots of 53BP1 co-immunoprecipitation. Right: quantification of the 53BP1 co-immunoprecipitation (n = 2). Data represent means ± SEM.


( G ) RT - qPCR validation of expression of FOXO3a , FOXO4 , PSMD11 ( n = 6 ) .

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(G) RT-qPCR validation of the expression levels of FOXO3a, FOXO4, and PSMD11 (n = 6).


( H ) FOXO3a - mediated transcriptional activity was measured with a luciferase reporter under control of a FOXO - response element promoter . HEK293T were transfected with shRNAs for control , LSD1 , UBE4B , or both LSD1 UBE4B , followed by transfection of luciferase reporter together with a constitutively active form of FOXO3a ( TM ) 72 - 96 h later ( n = 6 ) . Data represent means ± . numerical data used to make this figure can be found in S1 Data .

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(H) The FOXO3a-mediated transcriptional activity was measured with a luciferase reporter under the control of a FOXO-response element promoter. HEK293T cells were transfected with shRNAs for control, LSD1, UBE4B, or both LSD1 and UBE4B, followed by transfection of the luciferase reporter together with a constitutively active form of FOXO3a (TM) 72-96 h later (n = 6). Data represent means ± SEM. The numerical data used to make this figure can be found in S1 Data.


( A ) Increased of proteasome subunits upon UBE4B LSD1 double knockdown in HEK293T

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(A) Increased protein levels of proteasome subunits upon the UBE4B and LSD1 double knockdown in HEK293T cells


. ( B ) UBE4B LSD1 single or double knockdowns increase proteasomal degradation of a reporter substrate , Suc - LLVY - Luciferin , whose degradation is measured in a luciferase release . double knockdown shows synergistic proteasomal activation when compared with knockdowns ( n = 6 ) .

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. (B) The UBE4B and LSD1 single or double knockdowns increase the proteasomal degradation of a reporter substrate, Suc-LLVY-Luciferin, whose degradation is measured in a luciferase release assay. The double knockdown shows synergistic proteasomal activation when compared with the individual knockdowns (n = 6).


( C ) autophagy activity is significantly increased in with UBE4B LSD1 double knockdown . in vivo activity of ATG4B protease , which cleaves LC3 precursors , was quantified via a Gaussia luciferase release ( see S5D Fig . , S5E Fig . , Materials Methods for details ) . HCT116 were analyzed 48 h after initiation of knockdown .

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(C) The autophagy activity is significantly increased in cells with the UBE4B and LSD1 double knockdown. The in vivo activity of the ATG4B protease, which cleaves LC3 precursors, was quantified via a Gaussia luciferase release assay (see S5D Fig., S5E Fig., and Materials and Methods for details). The HCT116 cells were analyzed 48 h after the initiation of the knockdown.


( D ) of LC3 - II in HEK293T with UBE4B LSD1 double knockdown or mock control . were treated with or without 10 mM 3 - methyladenine ( 3 - MA ) for 48h before lysis . Western blots indicate an increase in LC3 ( top panels ) in double UBE4B LSD1 knockdown ( middle panels ) , while actin control is unchanged ( bottom panels ) . graph shows of LC3 - II as normalized to actin ( n = 3 ) . A one - way ANOVA with matched multiple comparisons Tukey correction was used for statistics . Data represent means ± . numerical data used to make this figure can be found in S1 Data .

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

(D) Quantification of LC3-II levels in HEK293T cells with the UBE4B and LSD1 double knockdown or the mock control. The cells were treated with or without 10 mM 3-methyladenine (3-MA) for 48h before lysis. Western blots indicate an increase in LC3 levels (top panels) in the double UBE4B and LSD1 knockdown cells (middle panels), while the actin control is unchanged (bottom panels). The graph shows the quantification of LC3-II levels as normalized to actin levels (n = 3). A one-way ANOVA test with matched multiple comparisons and Tukey correction was used for statistics. Data represent means ± SEM. The numerical data used to make this figure can be found in S1 Data.


( A ) p53 small molecule activators Tenovin - 1 CP - 31398 reduce of misfolded SOD1 , as determined by SOD1G85R solubility in HEK293 . Increasing concentrations of p53 activators significantly decrease of SOD1G85R but not endogenous WT SOD1 in western blots of both supernatant pellet fractions .

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

(A) p53 small molecule activators Tenovin-1 and CP-31398 reduce the levels of misfolded SOD1 proteins, as determined by the SOD1G85R solubility assay in HEK293 cells. Increasing concentrations of the p53 activators significantly decrease the levels of SOD1G85R but not the endogenous WT SOD1 proteins in western blots of both supernatant and pellet fractions.


( B ) A decrease in p53 as result of shRNA knockdown increases of SOD1G85R but not WT SOD1 in SOD1G85R aggregation , as shown by western blots of both supernatant ( S ) ( n = 2 ) pellet ( P ) ( n = 3 ) fractions .

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(B) A decrease in p53 as the result of shRNA knockdown increases the levels of SOD1G85R but not WT SOD1 proteins in the SOD1G85R aggregation assay, as shown by western blots of both supernatant (S) (n = 2) and pellet (P) (n = 3) fractions.


( C ) A complete absence of p53 increases accumulation of SOD1G85R mutant in p53 - / - HCT116 when compared with controls . Representative western blots ( left panels ) of SOD1G85R in supernatant lysates are shown . middle graph indicates of G85R to WT SOD1 in presence or absence of p53 with varying amounts of transfected mutant SOD1 . right graph panel shows same data as shown in middle panel , but normalized to average SOD1G85R level for each amount of transfected plasmid . Data represent means ± . numerical data used to make this figure can be found in S1 Data .

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(C) A complete absence of p53 increases the accumulation of SOD1G85R mutant proteins in p53-/- HCT116 cells when compared with controls. Representative western blots (left panels) and quantification of SOD1G85R levels in the supernatant lysates are shown. The middle graph indicates the ratio of G85R to WT SOD1 proteins in the presence or absence of p53 with varying amounts of transfected mutant SOD1. The right graph panel shows the same data as shown in the middle panel, but normalized to the average SOD1G85R level for each amount of the transfected plasmid. Data represent means ± SEM. The numerical data used to make this figure can be found in S1 Data.


( A ) Left : p53 knockdown reverses enhanced clearance of SOD1G85R conferred by UBE4B LSD1 double knockdown . Total amounts of shRNAs were adjusted to be equal with nontargeting CTRL shRNAs . Right : of insoluble aggregated SOD1G85R in pellet fractions from HEK293T transfected with control , double ( UBE4B / LSD1 ) , or triple ( UBE4B / LSD1 / p53 ) shRNAs ( n = 2 ) .

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(A) Left: p53 knockdown reverses the enhanced clearance of SOD1G85R proteins conferred by the UBE4B and LSD1 double knockdown. Total amounts of shRNAs were adjusted to be equal with nontargeting CTRL shRNAs. Right: quantification of insoluble aggregated SOD1G85R in pellet fractions from HEK293T cells transfected with control, double (UBE4B/LSD1), or triple (UBE4B/LSD1/p53) shRNAs (n = 2).


( B ) degenerative TDP - 43M337V eye phenotype is exacerbated by knockdown of Drosophila homolog of p53 ( p53 RNAi ) , or by overexpression of a dominant - negative p53 mutant ( p53 . R155H ) . Expression of p53 RNAi , p53 . R155H , TDP - 43M337V are driven by GMR - Gal4 .

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(B) The degenerative TDP-43M337V eye phenotype is exacerbated by the knockdown of the Drosophila homolog of p53 (p53 RNAi), or by the overexpression of a dominant-negative p53 mutant (p53.R155H). Expression of p53 RNAi, p53.R155H, and TDP-43M337V are driven by GMR-Gal4.


( C ) p53 - activating drug Tenovin - 1 ( TEN1 ) protects spinal cord motor neurons from SOD1G85R - induced proteotoxicity . Rat spinal cord cultures were grown as described in Materials Methods treated with 0 . 8 μM TEN1 or vehicle ( VEH ) DMSO , followed by infection of HSV - SOD1G85R or HSV - LacZ control after 24 h . At day 5 post - infection , were fixed stained with a motor neuron - specific anti - neurofilament H ( NF - H ) antibody . Left : representative of motor neurons in each condition . Scale bar = 50 μm . Right : of motor neuron survival ( one - way ANOVA with multiple comparisons Tukey correction ) . Data represent means ± .

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(C) The p53-activating drug Tenovin-1 (TEN1) protects spinal cord motor neurons from SOD1G85R-induced proteotoxicity. Rat spinal cord cultures were grown as described in Materials and Methods and treated with 0.8 μM TEN1 or vehicle (VEH) DMSO, followed by infection of HSV-SOD1G85R or the HSV-LacZ control after 24 h. At day 5 post-infection, cells were fixed and stained with a motor neuron-specific anti-neurofilament H (NF-H) antibody. Left: representative images of motor neurons in each condition. Scale bar = 50 μm. Right: quantification of motor neuron survival (one-way ANOVA with multiple comparisons and Tukey correction). Data represent means ± SEM.


( A ) Top hit selected candidates are listed . plot shows relative S9 . 6 values for transformed with each of indicated pool of siRNAs .

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(A) Top hit selected candidates are listed. The plot shows relative S9.6 nuclear intensity values for cells transformed with each of the indicated pool of siRNAs.


( B ) Representative of HeLa immunostained with S9 . 6 nucleolin antibodies after transfection with at least a pool of two siRNAs of each original pool .

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(B) Representative images of HeLa cells immunostained with S9.6 and nucleolin antibodies after transfection with at least a pool of two siRNAs of each original pool.


( C ) Relative S9 . 6 signal per nucleus after nucleolus signal removal in HeLa transfected with indicated siRNAs . More than 250 total from three independent experiments were considered . median of each population is shown . Boxes whiskers indicate 25 - 75 10 - 90 percentiles , respectively . * * * p < 0 . 001 ( Mann - Whitney U ) .

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(C) Relative S9.6 signal intensity per nucleus after nucleolus signal removal in HeLa cells transfected with the indicated siRNAs. More than 250 total cells from three independent experiments were considered. The median of each population is shown. Boxes and whiskers indicate 25-75 and 10-90 percentiles, respectively. ***p<0.001 (Mann-Whitney U test).


( A ) Relative DRIP - qPCR signal values at RPL13A , APOE , MIB2 RHOT2 genes in HeLa transfected with indicated siRNAs treated in vitro with RNase H pre - immunoprecipitation where indicated . mean ± from at least three independent experiments is shown . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( one - paired t - ) . Black stars denote significant increases whereas red stars denote significant decreases .

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(A) Relative DRIP-qPCR signal values at RPL13A, APOE, MIB2 and RHOT2 genes in HeLa cells transfected with the indicated siRNAs and treated in vitro with RNase H pre-immunoprecipitation where indicated. The mean ± SEM from at least three independent experiments is shown. *p<0.05, **p<0.01, ***p<0.001 (one-tailed paired t-test). Black stars denote significant increases whereas red stars denote significant decreases.


( B ) Representative of immunostaining with S9 . 6 anti - nucleolin antibodies in HeLa transfected with indicated

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(B) Representative images of immunostaining with S9.6 and anti-nucleolin antibodies in HeLa cells transfected with the indicated siRNAs


( C ) Relative S9 . 6 signal per nucleus after nucleolus signal removal in HeLa after cytoplasm pre - extraction ( CE ) treated in vitro with RNaseIII RNase H where indicated . More than 500 total from three independent experiments were considered . median of each population is shown . Boxes whiskers indicate 25 - 75 10 - 90 percentiles , respectively . * * p < 0 . 01 , * * * p < 0 . 001 ( Mann - Whitney U ) .

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(C) Relative S9.6 signal intensity per nucleus after nucleolus signal removal in HeLa cells after cytoplasm pre-extraction (CE) and treated in vitro with RNaseIII and RNase H where indicated. More than 500 total cells from three independent experiments were considered. The median of each population is shown. Boxes and whiskers indicate 25-75 and 10-90 percentiles, respectively. **p<0.01, ***p<0.001 (Mann-Whitney U test).


( A ) Representative of single - alkaline gel electrophoresis ( comet ) of HeLa transfected with indicated siRNAs either pEGFP - C1 ( RNH1 - ) or pEGFP - M27 ( RNH1 + ) .

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(A) Representative images of single-cell alkaline gel electrophoresis (comet assay) of HeLa cells transfected with the indicated siRNAs and either pEGFP-C1 (RNH1-) or pEGFP-M27 (RNH1+).


( B ) Comet tail moment from single - alkaline gel electrophoresis ( comet ) of HeLa transfected with indicated siRNAs either pEGFP - C1 ( RNH1 - ) or pEGFP - M27 ( RNH1 + ) . More than 250 total were considered . mean ± of median from at least three independent experiments is shown , except for RAD1 , RAD17 UBE2B ( n = 2 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( one - unpaired t - ) . Black stars denote significant increases whereas red stars denote significant decreases .

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(B) Comet tail moment from single-cell alkaline gel electrophoresis (comet assay) of HeLa cells transfected with the indicated siRNAs and either pEGFP-C1 (RNH1-) or pEGFP-M27 (RNH1+). More than 250 total cells were considered. The mean ± SEM of the median from at least three independent experiments is shown, except for RAD1, RAD17 and UBE2B (n=2). *p<0.05, **p<0.01, ***p<0.001 (one-tailed unpaired t-test). Black stars denote significant increases whereas red stars denote significant decreases.


( A ) Representative of HeLa with more than 5 H3S10 - P foci after transfection with indicated siRNAs either pEGFP - C1 ( RNH1 - ) or pEGFP - M27 ( RNH1 + ) . Mitotic were excluded for by DAPI . More than 300 total were considered . Data represent mean ± from three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( one - paired t - ) . Black stars denote significant increases whereas red stars denote significant decreases .

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(A) Representative images and percentage of HeLa cells with more than 5 H3S10-P foci after transfection with the indicated siRNAs and either pEGFP-C1 (RNH1-) or pEGFP-M27 (RNH1+). Mitotic cells were excluded for the analysis by DAPI staining. More than 300 total cells were considered. Data represent mean ± SEM from three independent experiments. *p<0.05, **p<0.01, ***p<0.001 (one-tailed paired t-test). Black stars denote significant increases whereas red stars denote significant decreases.


( B ) Representative H3K9me2 signal of HeLa transfected with indicated siRNAs either pEGFP - C1 ( RNH1 - ) or pEGFP - M27 ( RNH1 + ) . At least three experiments were performed . median of each population in a representative experiment with at least 100 per condition is shown . Boxes whiskers indicate 25 - 75 10 - 90 percentiles , respectively . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( one - paired t - ) . Black stars denote significant increases whereas red stars denote significant decreases .

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(B) Representative images and H3K9me2 nuclear signal intensity of HeLa cells transfected with the indicated siRNAs and either pEGFP-C1 (RNH1-) or pEGFP-M27 (RNH1+). At least three experiments were performed. The median of each population in a representative experiment with at least 100 cells per condition is shown. Boxes and whiskers indicate 25-75 and 10-90 percentiles, respectively. *p<0.05, **p<0.01, ***p<0.001 (one-tailed paired t-test). Black stars denote significant increases whereas red stars denote significant decreases.


( A ) Fork velocity as measured by combing in HeLa transfected with indicated siRNAs either pEGFP - C1 ( RNH1 - ) or pEGFP - M27 ( RNH1 + ) . More than 200 tracks were considered except for RAD1 + RNH1 ( n = 125 ) , ATR + RNH1 ( n = 182 ) RAD18 + RNH1 ( n = 146 ) . Median values are indicated . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( Mann - Whitney U ) . Black stars denote significant increases whereas red stars denote significant decreases .

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(A) Fork velocity as measured by DNA combing assay in HeLa cells transfected with the indicated siRNAs and either pEGFP-C1 (RNH1-) or pEGFP-M27 (RNH1+). More than 200 tracks were considered except for RAD1 +RNH1 (n=125), ATR +RNH1 (n=182) and RAD18 +RNH1 (n=146). Median values are indicated. *p<0.05, **p<0.01, ***p<0.001 (Mann-Whitney U test). Black stars denote significant increases whereas red stars denote significant decreases.


( B ) Fork asymmetry as measured by combing in HeLa transfected with indicated siRNAs either pEGFP - C1 ( RNH1 - ) or pEGFP - M27 ( RNH1 + ) . From 40 to 200 total measurements were considered for each candidate . Median values are indicated . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( Mann - Whitney U ) . Black stars denote significant increases whereas red stars denote significant decreases .

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(B) Fork asymmetry as measured by DNA combing assay in HeLa cells transfected with the indicated siRNAs and either pEGFP-C1 (RNH1-) or pEGFP-M27 (RNH1+). From 40 to 200 total measurements were considered for each candidate. Median values are indicated. *p<0.05, **p<0.01, ***p<0.001 (Mann-Whitney U test). Black stars denote significant increases whereas red stars denote significant decreases.


( A ) Left column : Flow cytometry profiles showing content of indicated RNase III - treated . G1 ( red ) , S ( blue ) G2 ( green ) phases were calculated from profile . Right column : Flow cytometry histograms depicting of S9 . 6 signals in each phase of cycle for indicated . ( B ) of panel B . mean ± SD of S9 . 6 mean of five experiments is shown . * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 ( repeated measures ANOVA with Bonferroni ' s post - ) . ( C ) Graph represents fold change of S9 . 6 mean signal in G2 with respect to G1 . Values for five independent experiments together with mean are shown . * p < 0 . 05 ( one - paired t - ) .

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(A) Left column: Flow cytometry profiles showing DNA content of the indicated RNase III-treated cells. G1 (red), S (blue) and G2 (green) phases were calculated from the profile. Right column: Flow cytometry histograms depicting intensity of S9.6 signals in each phase of the cell cycle for the indicated cells. (B) Quantification of panel B. The mean ± SD of the S9.6 mean intensity of five experiments is shown. ***p<0.001, **p<0.01, *p<0.05 (repeated measures ANOVA test with Bonferroni's post-test). (C) Graph represents fold change of S9.6 mean signal in G2 with respect to G1 cells. Values for five independent experiments together with the mean are shown. *p<0.05 (one-tailed paired t-test).


( A ) - hybrids , damage fork stalling in 9 - 1 - 1 / ATR / CHK1 , ATM / CHK2 PRR deficient . - hybrids represent mean ± of DRIP data * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 ( Paired t - ) . damage represents mean of medians ± of comet tail moment data * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 ( paired t - ) . Fork stalling represents median ± of fork asymmetry data * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 ( Mann - Whitney U ) . Black stars denote significant increases whereas red stars denote significant decreases .

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(A) DNA-RNA hybrids, DNA damage and fork stalling in 9-1-1/ATR/CHK1, ATM/CHK2 and PRR deficient cells. DNA-RNA hybrids represent the mean ± SEM of all the DRIP data ***p<0.001, **p<0.01, *p<0.05 (Paired t-test). DNA damage represents the mean of medians ± SEM of all the comet tail moment data ***p<0.001, **p<0.01, *p<0.05 (paired t-test). Fork stalling represents the median ± SEM of all the fork asymmetry data ***p<0.001, **p<0.01, *p<0.05 (Mann-Whitney U test). Black stars denote significant increases whereas red stars denote significant decreases.


IF by confocal microscopy performed on KRAB - HA overexpressing . was performed with anti - HA ( Alexa - 488 , green ) , anti - HSPA5 ( Alexa - 647 , red ) was stained with Dapi ( blue ) .

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IF by confocal microscopy performed on KRAB-HA overexpressing cells. Staining was performed with anti-HA (Alexa-488, green), anti-HSPA5 (Alexa-647, red) and DNA was stained with Dapi (blue).


HA immunoprecipitation of stably expressed ZNF282 - HA in previously transfected with ΔDUF3669 - ZNF398 - GFP WT - ZNF398 - GFP followed by detection of ZNF398 constructs in IPs through Western - Blot using an anti - GFP antibody . Bottom : Western - Blot using an anti - HA antibody on IPs . Input = lysate , IP =

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HA immunoprecipitation of stably expressed ZNF282-HA in cells previously transfected with ΔDUF3669-ZNF398-GFP and WT-ZNF398-GFP followed by detection of ZNF398 constructs in the IPs through Western-Blot using an anti-GFP antibody. Bottom: Western-Blot using an anti-HA antibody on the IPs. Input = Cellular lysate, IP = Immunoprecipitate


Left : DUF3669 - only KRAB domain constructs used . Right : HA immunoprecipitation in previously co - transfected with ZNF398 - DUF3669 - HA either ZNF282 - KRAB - Flag or ZNF282 - DUF3669 - Flag followed by detection of either of these constructs in IPs through Western - Blot using an anti - Flag antibody . Bottom : Western - Blot using an anti - HA antibody on IPs .

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Left: DUF3669-only and KRAB domain constructs used. Right: HA immunoprecipitation in cells previously co-transfected with ZNF398-DUF3669-HA and either ZNF282-KRAB-Flag or ZNF282-DUF3669-Flag followed by detection of either of these protein constructs in the IPs through Western-Blot using an anti-Flag antibody. Bottom: Western-Blot using an anti-HA antibody on the IPs.


Histograms representing of KZFPs ( identified in Fig . 3B ) binding sites falling in TEs ( red ) or TSSs ( blue ) .

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Histograms representing the percentage of KZFPs (identified in Fig. 3B) binding sites falling in TEs (red) or TSSs (blue).


KZFPs binding sites on L1PA5 L1PA6 . Top : boxes containing interactors of KZFPs found enriched on L1PA5 , 6 . Middle : plot showing average ChIP - exo signals ( scaled between 0 1 ) for each selected KZFP plotted on top of L1PA5 L1PA6 multiple alignment ( MSA ) . Bottom : schematic representation of L1PA5 , 6 different domains .

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KZFPs binding sites on L1PA5 and L1PA6. Top: boxes containing the interactors of KZFPs found enriched on L1PA5,6. Middle: plot showing the average ChIP-exo signals (scaled between 0 and 1) for each selected KZFP plotted on top of L1PA5 and L1PA6 multiple sequence alignment (MSA). Bottom: schematic representation of L1PA5,6 different domains.


Human KZFPs KRAB domain A - boxes phylogenetic tree associated with their corresponding acid sequences colored according to Clustal ZAPPO color scheme ( residues sharing common physicochemical properties display same color : http : / / www . jal - view . org / help / html / colourSchemes / zappo . html ) . This figure also displays a zoom - in on variant KRAB domain cluster ( right ) . On this zoom - in , tested vKZFPs , whose interactomes were defined in our study , were marked by an asterisk . D . Box plot representing KAP1FC values in function of KZFPs . On a superimposed swarm plot , vKZFPs corresponding KAP1FC values were represented by red dots for their sKZFPs counterparts , by blue dots . Mann - Whitney two - sided rank .

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Human KZFPs KRAB domain A-boxes protein phylogenetic tree associated with their corresponding amino acid sequences colored according to the Clustal ZAPPO color scheme (residues sharing common physicochemical properties display the same color: http://www.jal- view.org/ help/html/colourSchemes/zappo.html). This figure also displays a zoom-in on the variant KRAB domain cluster (right). On this zoom-in, the tested vKZFPs, whose interactomes were defined in our study, were marked by an asterisk. D. Box plot representing KAP1FC values in function of KZFPs age. On a superimposed swarm plot, the individual vKZFPs corresponding KAP1FC values were represented by red dots and for their sKZFPs counterparts, by blue dots. Mann-Whitney two-sided rank test.


Immunoprecipitations of HA - tagged KRAB domains in order to check interaction with cKAP1 . Co - transfection of Flag - tagged cKAP1 ( upper panel ) or Flag - tagged hKAP1 ( lower panel ) HA - tagged ZNF398 DUF3669 domain negative control , H2ZYM4 M3XKG1 cKRAB domains ZNF93 hKRAB domain in KAP1 KO HAP1 followed by HA - immunoprecipitation . cKAP1 hKAP1 presence was revealed by Western - Blot using an anti - Flag antibody . Input = lysate , IP = Immunoprecipitate . Western - Blot using an HA antibody on IPs at bottom .

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Immunoprecipitations of HA-tagged KRAB domains in order to check
 interaction with cKAP1. Co-transfection of Flag-tagged cKAP1 (upper panel) or Flag-tagged hKAP1 (lower panel) and HA-tagged ZNF398 DUF3669 domain negative control, H2ZYM4 and M3XKG1 cKRAB domains and ZNF93 hKRAB domain in KAP1 KO HAP1 cells followed by HA-immunoprecipitation. cKAP1 and hKAP1 presence was revealed by Western-Blot using an anti-Flag antibody. Input = Cellular lysate, IP = Immunoprecipitate. Western-Blot using an HA antibody on the IPs at the bottom.


Immunoblot of mouse intestinal crypts treated with Wnt3a ( 60 ng / mL ) for indicated time . relative level was quantified shown under each band .

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Immunoblot analysis of mouse intestinal crypts treated with Wnt3a (60 ng/mL) for indicated time. The relative protein level was quantified and shown under each band.


Polysome profiles of mouse intestinal crypt treated with or without Wnt3a for 30 min . right panels show distributions of Ythdf1 Actb in polysome fractions . Data are represented as mean ± . * p < 0 . 05 , * * p < 0 . 01 ( 3 biological replicates , t - ) .

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Polysome profiles of mouse intestinal crypt treated with or without Wnt3a for 30 min. The right panels show the distributions of Ythdf1 and Actb in polysome fractions. Data are represented as mean ± SEM. *p<0. 05, **p<0. 01 (3 biological replicates, t-test).


Immunoblot of SW620 with or without APC overexpression . relative level was quantified shown under each band .

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Immunoblot analysis of SW620 cells with or without APC overexpression. The relative protein level was quantified and shown under each band.


Polysome profiles of SW620 with or without APC overexpression . right panels show distributions of YTHDF1 ACTB in polysome fractions . Data are represented as mean ± . * p < 0 . 05 , * * p < 0 . 01 ( 3 biological replicates , t - ) .

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Polysome profiles of SW620 cells with or without APC overexpression. The right panels show the distributions of YTHDF1 and ACTB in polysome fractions. Data are represented as mean ± SEM. *p<0. 05, **p<0. 01 (3 biological replicates, t-test).


Dual - Luciferase wih a construct bearing 5 ' UTR of YTHDF1 in SW620 with or without APC overexpression . Data are represented as mean ± . * p < 0 . 05 , * * p < 0 . 01 ( 3 biological replicates , t - ) .

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Dual-Luciferase assay wih a construct bearing the 5'UTR of YTHDF1 in SW620 cells with or without APC overexpression. Data are represented as mean ± SEM. *p<0. 05, **p<0. 01 (3 biological replicates, t-test).


YTHDF1 expression in intestinal tissue from 4 pairs of Apc + / + Apcmin / + mice . N , normal tissue . T , tissue . A , adjacent tissue .

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YTHDF1 protein expression in intestinal tissue from 4 pairs of Apc+/+ and Apcmin/+ mice. N, normal tissue. T, tumor tissue. A, tumor adjacent tissue.


YTHDF1 in matched samples of human CRC tissue adjacent non - tissue . Scale bar , 450 μm . right panel shows statistics of scores . Chi - square ; n = 75 for each group .

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YTHDF1 staining in matched samples of human CRC tissue and adjacent non-tumor tissue. Scale bar, 450 μm. The right panel shows the statistics of IHC scores. Chi-square test; n = 75 for each group.


representative jejunum from wildtype ( Ythdf1CTL ) Ythdf1cKO mice . Left : hematoxylin eosin ( H & E ) ; middle : BrdU ; right : of crypt height BrdU + . Scale bar , 50 μm . Data are represented as mean ± . ( 9 biological replicates , t - ) .

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The representative jejunum from wildtype (Ythdf1CTL) and Ythdf1cKO mice. Left: hematoxylin and eosin staining (H&E); middle: BrdU staining; right: quantification of crypt height and BrdU+ cells. Scale bar, 50 μm. Data are represented as mean ± SEM. (9 biological replicates, t-test).


Immunoblot of small intestines from Ythdf1CTL Ythdf1cKO mice after 12 Gy IR .

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Immunoblot analysis of small intestines from Ythdf1CTL and Ythdf1cKO mice after 12 Gy IR.


YTHDF1 ( left ) Ki67 ( right ) of small intestine from wildtype mice 72 hr after 12 Gy IR . Scale bar , 50 μm . Red arrows indicate proliferating .

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YTHDF1 (left) and Ki67 (right) staining of small intestine from wildtype mice 72 hr after 12 Gy IR. Scale bar, 50 μm. Red arrows indicate the proliferating cells.


Small intestines from Ythdf1CTL Ythdf1cKO mice 72 hr after 12 Gy IR . Left : H & E ; middle : Ki67 ; right : of crypt height Ki67 + . Scale bar , 50 μm . Vertical bars indicate of crypts . Data are represented as mean ± . * * * * p < 0 . 0001 ( 9 biological replicates , t - ) .

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Small intestines from Ythdf1CTL and Ythdf1cKO mice 72 hr after 12 Gy IR. Left: H&E staining; middle: Ki67 staining; right: quantification of crypt height and Ki67+ cells. Scale bar, 50 μm. Vertical bars indicate the length of the crypts. Data are represented as mean ± SEM. ****p<0. 0001 (9 biological replicates, t-test).


RT - qPCR of small intestines from Ythdf1CTL Ythdf1cKO mice 72 hr after 12 Gy IR . Data are represented as mean ± . * p < 0 . 05 , * * p < 0 . 01 ( 5 biological replicates , t - ) .

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RT-qPCR analysis of small intestines from Ythdf1CTL and Ythdf1cKO mice 72 hr after 12 Gy IR. Data are represented as mean ± SEM. *p<0. 05, **p<0. 01 (5 biological replicates, t-test).


Small intestines from 24 - week - old Apcmin / + ; Ythdf1CTL Apcmin / + ; Ythdf1cKO mice .

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Small intestines from 24-week-old Apcmin/+; Ythdf1CTL and Apcmin/+; Ythdf1cKO mice.


H & E of small intestines from 24 - week - old Apcmin / + ; Ythdf1CTL Apcmin / + ; Ythdf1cKO mice . Scale bar , 1 mm . black arrows indicate tumors .

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H&E staining of small intestines from 24-week-old Apcmin/+; Ythdf1CTL and Apcmin/+; Ythdf1cKO mice. Scale bar, 1 mm. The black arrows indicate the tumors.


number ( C ) distribution ( D ) from small intestines of 24 - week - old Apcmin / + ; Ythdf1CTL Apcmin / + ; Ythdf1cKO mice . Data are shown as mean ± ( 6 mice for each group ) . * * p < 0 . 01 , * * * p < 0 . 001 ( t - ) .

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Tumor number (C) and size distribution (D) from the small intestines of 24-week-old Apcmin/+; Ythdf1CTL and Apcmin/+; Ythdf1cKO mice. Data are shown as mean ± SEM (6 mice for each group). **p<0. 01, ***p<0. 001 (t-test).


Colons from Ythdf1CTL Ythdf1cKO mice on day 84 of AOM / DSS induction .

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Colons from Ythdf1CTL and Ythdf1cKO mice on day 84 of AOM/DSS induction.


H & E of colons from Apcmin / + ; Ythdf1CTL Apcmin / + ; Ythdf1cKO mice after AOM / DSS induction . Scale bar , 200 μm .

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H&E staining of colons from Apcmin/+; Ythdf1CTL and Apcmin/+; Ythdf1cKO mice after AOM/DSS induction. Scale bar, 200 μm.


Colon number ( H ) distribution ( I ) from Ythdf1CTL Ythdf1cKO mice on day 84 of induction . Data are shown as mean ± ( 6 mice for each group ) . * * p < 0 . 01 ( t - ) .

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Colon tumor number (H) and size distribution (I) from Ythdf1CTL and Ythdf1cKO mice on day 84 of induction. Data are shown as mean ± SEM (6 mice for each group). **p<0. 01 (t-test).


in situ hybridization for Ythdf1 mRNA in Ythdf1CTL Ythdf1cKO small intestines . Scale bar , 50 μm .

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Fluorescent in situ hybridization for Ythdf1 mRNA in Ythdf1CTL and Ythdf1cKO small intestines. Scale bar, 50 μm.


Immunoblot of fractionated murine intestine enriched for villus crypt .

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Immunoblot analysis of fractionated murine intestine enriched for villus cells and crypt cells.


Flowcytometry of YTHDF1 expression in GFPhigh / low / neg populations from Lgr5 - GFP - IRES - creERT2 mice .

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Flowcytometry analysis of YTHDF1 protein expression in GFPhigh/low/neg populations from Lgr5-GFP-IRES-creERT2 mice.


of organoids from Lgr5 - creERT2 : Ythdf1fl / fl mice in Wnt3a - conditioned medium without or with 4 - OHT induction infected with lentivirus expressing YTHDF1 or YTHDF1 mutant . Scale bar , 250 μm .

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Morphology of organoids from Lgr5-creERT2:Ythdf1fl/fl mice in Wnt3a-conditioned medium without or with 4-OHT induction and infected with lentivirus expressing YTHDF1 or YTHDF1 mutant. Scale bar, 250 μm.


of differentiated versus undifferentiated organoids from ( D ) . Data are represented as mean ± . * * p < 0 . 01 ( 4 biological replicates , t - ) .

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Quantification of differentiated versus undifferentiated organoids from (D). Data are represented as mean ± SEM. **p<0. 01 (4 biological replicates, t-test).


Relative mRNA of intestinal differentiation marker genes in organoids described in ( D ) . Data are represented as mean ± . 4 biological replicates . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( t - ) .

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Relative mRNA levels of intestinal stem cell and differentiation marker genes in organoids described in (D). Data are represented as mean ± SEM. 4 biological replicates. *p<0. 05, **p<0. 01, ***p<0. 001 (t-test).


Scatterplot of mRNA expression ribosome profiling data from control YTHDF1 knockdown . upregulated ( red ) downregulated ( blue ) genes in translational efficiency ( TE ) are highlighted .

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Scatterplot of mRNA expression and ribosome profiling data from control and YTHDF1 knockdown cells. The upregulated (red) and downregulated (blue) genes in translational efficiency (TE) are highlighted.


relative m6A peak coverage in control for transcripts grouped according to TE changes after YTHDF1 knockdown . POI : peak over input . Down , genes significantly downregulated in YTHDF1 knockdown ( < 0 . 5 fold ) . Unchanged , genes not significantly changed . Up , genes significantly upregulated ( > 2 fold ) . upper lower quartiles median are shown for each group . Mann - Whitney . * * p < 0 . 01 . * * * p < 0 . 001 .

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The relative m6A peak coverage in control cells for transcripts grouped according to TE changes after YTHDF1 knockdown. POI: peak over input. Down, genes significantly downregulated in YTHDF1 knockdown cells (<0.5 fold). Unchanged, genes not significantly changed. Up, genes significantly upregulated (>2 fold). The upper and lower quartiles and the median are shown for each group. Mann-Whitney test. **p<0. 01. ***p<0. 001.


Violin plots showing TE change between control YTHDF1 knockdown for not - methylated ( non - m6A ) methylated ( m6A ) transcripts . upper lower quartiles median are indicated for each group . Mann - Whitney .

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Violin plots showing TE change between control and YTHDF1 knockdown cells for not-methylated (non-m6A) and methylated (m6A) transcripts. The upper and lower quartiles and the median are indicated for each group. Mann-Whitney test.


Cumulative distributions of TE change for non - m6A m6A transcripts as in ( C ) . Kolmogorov - Smirnov .

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Cumulative distributions of TE change for non-m6A and m6A transcripts as in (C). Kolmogorov-Smirnov test.


showing TE of Wnt signaling components in control YTHDF1 knockdown . values show fold change of indicated color of .

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Heatmap showing the TE of Wnt signaling components in control and YTHDF1 knockdown cells. The values show the fold change of the indicated color of the heatmap.


Immunoblot of HCT116 with YTHDF1 knockdown . Two major bands were detected for human TCF7L2 due to alternative splicing .

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Immunoblot analysis of HCT116 cells with YTHDF1 knockdown. Two major bands were detected for human TCF7L2 due to alternative splicing.


Polysome profiles of HCT116 with or without YTHDF1 knockdown . right panels show distributions of TCF7L2 , ACTB in polysome fractions . Data are represented as mean ± . * p < 0 . 05 , * * p < 0 . 01 ( 3 biological replicates , t - ) .

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Polysome profiles of HCT116 cells with or without YTHDF1 knockdown. The right panels show the distributions of TCF7L2, and ACTB in polysome fractions. Data are represented as mean ± SEM. *p<0. 05, **p<0. 01 (3 biological replicates, t-test).


Immunoblot of crypts from Ythdf1CTL Ythdf1cKO mice . Multiple bands were detected for mouse TCF7L2 representing different isoforms .

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Immunoblot analysis of crypts from Ythdf1CTL and Ythdf1cKO mice. Multiple bands were detected for mouse TCF7L2 representing different isoforms.


Immunoblot of mouse intestinal crypts treated with Wnt3a for 60 min .

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Immunoblot analysis of mouse intestinal crypts treated with Wnt3a for 60 min.


MeRIP - qPCR of m6A of TCF7L2 in crypts treated with or without Wnt3a . Data are represented as mean ± . * p < 0 . 05 ( 3 biological replicates , t - ) .

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MeRIP-qPCR analysis of m6A levels of TCF7L2 in crypts treated with or without Wnt3a. Data are represented as mean ± SEM. *p<0. 05 (3 biological replicates, t-test).


RIP of interaction of YTHDF1 with TCF7L2 mRNA . enrichment was measured by qPCR normalized to input . Data are represented as mean ± . * p < 0 . 05 ( 3 biological replicates , t - ) .

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RIP analysis of the interaction of YTHDF1 with TCF7L2 mRNA. The enrichment was measured by qPCR and normalized to input. Data are represented as mean ± SEM. *p<0. 05 (3 biological replicates, t-test).


β - catenin / TCF4 reporter activity in YTHDF1 knockdown infected with lentivirus expressing TCF7L2 . Data are represented as mean ± . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( 3 biological replicates , t - ) .

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β-catenin/TCF4 reporter activity in YTHDF1 knockdown cells infected with lentivirus expressing TCF7L2. Data are represented as mean ± SEM. *p<0. 05, **p<0. 01, ***p<0.001 (3 biological replicates, t-test).


of organoids from Lgr5 - creERT2 : Ythdf1fl / fl mice in Wnt3a - conditioned medium without or with 4 - OHT induction infected with lentivirus expressing TCF7L2 . Scale bar , 250 μm . of differentiated versus undifferentiated organoids from ( H ) . Data are represented as mean ± . * * p < 0 . 01 , * * * p < 0 . 001 ( 3 biological replicates , t - ) .

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Morphology of organoids from Lgr5-creERT2:Ythdf1fl/fl mice in Wnt3a-conditioned medium without or with 4-OHT induction and infected with lentivirus expressing TCF7L2. Scale bar, 250 μm. Quantification of differentiated versus undifferentiated organoids from (H). Data are represented as mean ± SEM. **p<0. 01, ***p<0. 001 (3 biological replicates, t-test).


Colons from mice in ( A ) treated with or without TAM on day 80 of AOM / DSS induction .

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Colons from mice in (A) treated with or without TAM on day 80 of AOM/DSS induction.


Colon number from mice treated with or without TAM on day 80 of AOM / DSS induction . Data are shown as mean ± ( 7 mice for each group ) . * * * * p < 0 . 0001 ( t - ) .

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Colon tumor number from mice treated with or without TAM on day 80 of AOM/DSS induction. Data are shown as mean ± SEM (7 mice for each group). ****p<0. 0001 (t-test).


Immunoblot of 2 pairs of colon tumors from mice treated with or without TAM .

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Immunoblot analysis of 2 pairs of colon tumors from mice treated with or without TAM.


Kaplan - Meier survival plots for Apcmin / + ; Lgr5 - creERT2 : Ythdf1fl / fl mice ( 8 mice for each group ) treated with or without TAM . Log - rank ( Mantel Cox ) . * * p < 0 . 01 ( t - ) .

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Kaplan-Meier survival plots for Apcmin/+; Lgr5-creERT2:Ythdf1fl/fl mice (8 mice for each group) treated with or without TAM. Log-rank (Mantel Cox) test. **p<0. 01 (t-test).


( A ) Motility of C . c . ΔpseI expressing predicted Leg or Pse synthases ( LegI or PseI , in yellow or red , respectively ) from Plac on pSRK - Gm . Overnight cultures were spotted on PYE soft ( 0 . 3 % ) agar plates with gentamycin IPTG ( 0 . 5 mM ) incubated for 3 days at 30 ° C . Only predicted PseI - like synthase coding sequences ( CDSs ) restore motility of C . c . ΔpseI . Note that predicted synthases labeled in do not share similarities with LegI or PseI of C . jejuni . Motility assays are representative of three independent experiments .

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(A) Motility assay of C.c. ΔpseI cells expressing predicted Leg or Pse synthases (LegI or PseI, in yellow or red, respectively) from Plac on pSRK-Gm. Overnight cultures were spotted on PYE soft (0.3%) agar plates with gentamycin and IPTG (0.5 mM) and incubated for 3 days at 30°C. Only predicted PseI-like synthase coding sequences (CDSs) restore motility of the C.c. ΔpseI cells. Note that the predicted synthases labeled in white do not share similarities with LegI or PseI of C. jejuni. Motility assays are representative of three independent experiments.


( B ) Immunoblot of extracts from in ( A ) probed with antibodies to C . crescentus FljK ( FljKCc ) . migration of flagellins in ΔpseI is shifted towards lower molecular mass suggesting that post - translational modification of flagellin is defective in ΔpseI mutant . Molecular sizes are indicated by blues lines ( in kDa ) . Bs : Brevundimonas subvibrioides , Bl : Brevundimonas lutea , Bv : Brevundimonas viscosa , Dv : Dermabacter vaginalis , Li : Leptospira interrogans , Tp : Treponema pallidum , Td : Treponema denticola , AbACICU : Acinetobacter baumannii ACICU , AbLUH : Acinetobacter baumannii LUH , AbLAC : Acinetobacter baumannii LAC - 4 , Lp : Legionella pneumophila , PaY82 : Pseudomonas aeruginosa Y82 , PaPAPS475 : Pseudomonas aeruginosa PAPS475 , Sj : Shewanella japonicum , Basu : Bacillus subtilis , Ks : Kurthia sibirica , Cb : Clostridium botulinum , Gk : Geobacillus kaustophilus , Mh : Moorella humiferrea , Myco : Mycobacterium sp . KS0706 , HrrPV6 : Halorubrum sp . PV6 , Ms : Methanobrevibacter smithii . genes come from synthetic fragments codon optimized for E . coli ( except synthase CDS from Gk that is codon optimized for C . c . ) . Empty carets indicate position of modified ( glycosylated ) flagellin , whereas filled carets mark unmodified flagellin . Yellow boxes indicate PseI functional orthologs . Immunoblots are representative of at least two independent experiments .

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(B) Immunoblot of cell extracts from cells in (A) probed with antibodies to C. crescentus FljK (FljKCc). The migration of flagellins in ΔpseI cells is shifted towards lower molecular mass suggesting that post-translational modification of flagellin is defective in ΔpseI mutant. Molecular sizes are indicated by the blues lines (in kDa). Bs: Brevundimonas subvibrioides, Bl: Brevundimonas lutea, Bv: Brevundimonas viscosa, Dv: Dermabacter vaginalis, Li: Leptospira interrogans, Tp: Treponema pallidum, Td: Treponema denticola, AbACICU: Acinetobacter baumannii ACICU, AbLUH: Acinetobacter baumannii LUH, AbLAC: Acinetobacter baumannii LAC-4, Lp: Legionella pneumophila, PaY82: Pseudomonas aeruginosa Y82, PaPAPS475: Pseudomonas aeruginosa PAPS475, Sj: Shewanella japonicum, Basu: Bacillus subtilis, Ks: Kurthia sibirica, Cb: Clostridium botulinum, Gk: Geobacillus kaustophilus, Mh: Moorella humiferrea, Myco: Mycobacterium sp. KS0706, HrrPV6: Halorubrum sp. PV6, Ms: Methanobrevibacter smithii. All the genes come from synthetic fragments codon optimized for E. coli (except the synthase CDS from Gk that is codon optimized for C.c.). Empty carets indicate the position of modified (glycosylated) flagellin, whereas filled carets mark unmodified flagellin. Yellow boxes indicate PseI functional orthologs. Immunoblots are representative of at least two independent experiments.


( A ) ( B ) Motility assays of WT , ΔlegI ( A ) ΔflmG ( B ) B . s . . Overnight cultures of WT mutants harboring empty pSRK - Gm vector ( + Plac ) or corresponding complementing plasmid were spotted on PYE soft agar plates with gentamycin IPTG ( 0 . 5 mM ) incubated for 7 days at 30 ° C . Motility assays are representative of three independent experiments . same original picture was used to generate Figs 3B EV2C .

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(A) and (B) Motility assays of WT, ΔlegI (A) and ΔflmG (B) B.s. cells. Overnight cultures of WT and mutants harboring the empty pSRK-Gm vector (+Plac) or the corresponding complementing plasmid were spotted on PYE soft agar plates with gentamycin and IPTG (0.5 mM) and incubated for 7 days at 30°C. Motility assays are representative of three independent experiments. The same original picture was used to generate Figs 3B and EV2C.


( C ) ( D ) Immunoblots of extracts ( ) supernatants ( SN ) of cultures from WT mutant probed with antibodies to FljKCc , that detect B . s . flagellins . estimated molecular mass ( in kDa ) are indicated by blue lines on left . Empty carets indicate position of modified ( glycosylated ) flagellin , whereas filled carets mark unmodified flagellin . Immunoblots are representative of at least two independent experiments .

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(C) and (D) Immunoblots of cell extracts (cells) and supernatants (SN) of cultures from WT and mutant cells probed with antibodies to FljKCc, that detect the B.s. flagellins. The estimated molecular mass (in kDa) are indicated by the blue lines on the left. Empty carets indicate the position of modified (glycosylated) flagellin, whereas filled carets mark unmodified flagellin. Immunoblots are representative of at least two independent experiments.


( F ) Flagellum measurements determined by of WT mutants B . s . : WT , ΔlegX ( ΔBresu _ 3267 ) , ΔlegB ( ΔBresu _ 3266 ) expressing legX in trans from Plac on a plasmid , ΔlegH ( ΔBresu _ 0506 ) , ΔlegI ( ΔBresu _ 0507 ) , ΔflmG ( ΔBresu _ 2406 ) ΔlegI ΔflmG . Box plots represent distribution of flagellum lengths . cross indicates average , central band indicates median , upper lower sides of box indicate upper lower quartiles , respectively , whiskers indicate minimum maximum measured . Twenty - five independent flagella were measured different ( biological replicates ) for each from .

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(F) Flagellum length measurements determined by TEM of WT and mutants B.s. cells: WT, ΔlegX (ΔBresu_3267), ΔlegB (ΔBresu_3266) expressing legX in trans from Plac on a plasmid, ΔlegH (ΔBresu_0506), ΔlegI (ΔBresu_0507), ΔflmG (ΔBresu_2406) and ΔlegI ΔflmG. Box plots represent the distribution of the flagellum lengths. The cross indicates the average length, the central band indicates the median, the upper and lower sides of the box indicate the upper and lower quartiles, respectively, and the whiskers indicate the minimum and the maximum length measured. Twenty-five independent flagella were measured different cells (biological replicates) for each strain from TEM images.


( A ) Motility assays of B . s . ΔlegI complemented with LegI - type ( yellow ) or PseI - type ( red ) synthases expressed from Plac on pSRK - Gm . Motility assays are representative of three independent experiments .

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(A) Motility assays of B.s. ΔlegI cells complemented with LegI-type (yellow) or PseI-type (red) synthases expressed from Plac on pSRK-Gm. Motility assays are representative of three independent experiments.


( B ) Immunoblots probed with antibodies to FljKCc , revealing extracts of flagellin in B . s . ΔlegI derivatives shown in ( A ) . blue lines represent migration of molecular standards ( in kDa ) . Bl : Brevundimonas lutea , Bv : Brevundimonas viscosa , Dv : Dermabacter vaginalis , Li : Leptospira interrogans , Tp : Treponema pallidum , Td : Treponema denticola , AbACICU : Acinetobacter baumannii ACICU , AbLUH : Acinetobacter baumannii LUH , AbLAC : Acinetobacter baumannii LAC - 4 , Lp : Legionella pneumophila , PaY82 : Pseudomonas aeruginosa Y82 , PaPAPS475 : Pseudomonas aeruginosa PAPS475 , Sj : Shewanella japonicum , Basu : Bacillus subtilis , Ks : Kurthia sibirica , Cb : Clostridium botulinum , Gk : Geobacillus kaustophilus , Mh : Moorella humiferrea , Myco : Mycobacterium sp . KS0706 , HrrPV6 : Halorubrum sp . PV6 , Ms : Methanobrevibacter smithii . genes come from synthetic fragments codon optimized for E . coli ( except synthase CDS from Gk that is codon optimized for C . c . ) . Empty carets indicate position of modified ( glycosylated ) flagellin , whereas filled carets mark unmodified flagellin . Red boxes indicate LegI functional orthologs . Immunoblots are representative of at least two independent experiments .

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

(B) Immunoblots probed with antibodies to FljKCc, revealing the cell extracts levels of flagellin in B.s. ΔlegI derivatives shown in (A). The blue lines represent the migration of the molecular size standards (in kDa). Bl: Brevundimonas lutea, Bv: Brevundimonas viscosa, Dv: Dermabacter vaginalis, Li: Leptospira interrogans, Tp: Treponema pallidum, Td: Treponema denticola, AbACICU: Acinetobacter baumannii ACICU, AbLUH: Acinetobacter baumannii LUH, AbLAC: Acinetobacter baumannii LAC-4, Lp: Legionella pneumophila, PaY82: Pseudomonas aeruginosa Y82, PaPAPS475: Pseudomonas aeruginosa PAPS475, Sj: Shewanella japonicum, Basu: Bacillus subtilis, Ks: Kurthia sibirica, Cb: Clostridium botulinum, Gk: Geobacillus kaustophilus, Mh: Moorella humiferrea, Myco: Mycobacterium sp. KS0706, HrrPV6: Halorubrum sp. PV6, Ms: Methanobrevibacter smithii. All the genes come from synthetic fragments codon optimized for E. coli (except the synthase CDS from Gk that is codon optimized for C.c.). Empty carets indicate the position of modified (glycosylated) flagellin, whereas filled carets mark unmodified flagellin. Red boxes indicate LegI functional orthologs. Immunoblots are representative of at least two independent experiments.


( A ) Motility assays of WT B . s . ΔBresu _ 3266 ( ΔlegB ) expressing Bresu _ 3266 ( LegB ) , Bresu _ 3267 ( LegX ) or Bresu _ 3266 - Bresu _ 3267 ( LegB - LegX , Bresu _ 3266 - 67 ) from Plac on plasmid pSRK - Gm . Motility assays are representative of three independent experiments . ( D ) Motility of WT B . s . ΔBresu _ 0506 ( ΔlegH ) complemented with a plasmid expressing Bresu _ 0506 ( LegH ) from Plac on pSRK - Gm . Motility assays are representative of three independent experiments .

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(A) Motility assays of WT B.s. and ΔBresu_3266 (ΔlegB) cells expressing Bresu_3266 (LegB), Bresu_3267 (LegX) or Bresu_3266-Bresu_3267 (LegB-LegX, Bresu_3266-67) from Plac on plasmid pSRK-Gm. Motility assays are representative of three independent experiments. (D) Motility assay of WT B.s. and ΔBresu_0506 (ΔlegH) cells complemented with a plasmid expressing Bresu_0506 (LegH) from Plac on pSRK-Gm. Motility assays are representative of three independent experiments.


( B ) Immunoblots probed with anti - FljKCc antibodies to reveal flagellins in extracts ( ) supernatants ( SN ) of strains described in ( A ) . ( E ) Immunoblots probed with anti - FljKCc antibodies to reveal flagellins in extracts ( ) supernatants ( SN ) of strains described in ( D ) . Immunoblots are representative of two independent experiments .

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(B) Immunoblots probed with anti-FljKCc antibodies to reveal flagellins in cell extracts (cells) and supernatants (SN) of the strains described in (A). (E) Immunoblots probed with anti-FljKCc antibodies to reveal flagellins in cell extracts (cells) and supernatants (SN) of the strains described in (D). Immunoblots are representative of two independent experiments.


( A ) Motility of ΔBresu _ 3267 ( ΔlegX ) B . s . compared to WT B . s . harbouring empty pSRK - Gm vector ( + Plac ) or a complementing derivative with PtmE / LegX orthologs from C . jejuni ( Cj ) , Tanerella forsythia ( Tf ) or P . aeruginosa NMI1897 ( PaNMI ) . Motility assays are representative of three independent experiments .

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(A) Motility assay of ΔBresu_3267 (ΔlegX) B.s. cells compared to WT B.s. cells harbouring the empty pSRK-Gm vector (+Plac) or a complementing derivative with PtmE/LegX orthologs from C. jejuni (Cj), Tanerella forsythia (Tf) or P. aeruginosa NMI1897 (PaNMI). Motility assays are representative of three independent experiments.


( B ) Immunoblots probed with anti - FljKCc antibodies from lysates ( ) supernatants ( SN ) of WT B . s . ΔBresu _ 3267 ( ΔlegX ) cultures harbouring either empty pSRK - Gm vector ( + Plac ) or a complementing plasmid expressing different PtmE / LegX orthologs from C . jejuni ( Cj ) , Tanerella forsythia ( Tf ) or P . aeruginosa NMI1897 ( PaNMI ) . Molecular standards are indicated by blue lines with corresponding value in kDa . Empty carets indicate position of modified ( glycosylated ) flagellin , whereas filled carets mark unmodified flagellin . Immunoblots are representative of two independent experiments .

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

(B) Immunoblots probed with anti-FljKCc antibodies from cell lysates (cells) and supernatants (SN) of WT B.s. and ΔBresu_3267 (ΔlegX) cultures harbouring either the empty pSRK-Gm vector (+Plac) or a complementing plasmid expressing different PtmE/LegX orthologs from C. jejuni (Cj), Tanerella forsythia (Tf) or P. aeruginosa NMI1897 (PaNMI). Molecular size standards are indicated by the blue lines with the corresponding value in kDa. Empty carets indicate the position of modified (glycosylated) flagellin, whereas filled carets mark unmodified flagellin. Immunoblots are representative of two independent experiments.


( B ) Anti - FljKCc immunoblot analyses of whole lysates ( ) from C . crescentus ( C . c . ) mutant cultures expressing fljKBssyn ( codon optimized for E . coli ) flmGBs from replicative pMT463 plasmid , in presence or absence of a compatible integrative plasmid carrying a legBs synthetic operon ( pXGFP4 - legBs ) in presence or absence of an additional compatible replicative plasmid carrying Bresu _ 3267 ( pMT375 ) . In presence of pXGFP4 - legBs ( integrated at xylX locus ) pMT375 - Bresu _ 3267 , FljKBssyn migration is shifted toward higher molecular mass , indicative of glycosylation . Note that in C . c . Δfljx6 background , six flagellin - encoding genes have been deleted detected by antibodies only corresponds to FljKBssyn . legBs synthetic operon ( in red ) is composed of Bresu _ 3266 , Bresu _ 0765 , Bresu _ 0506 , Bresu _ 3264 , Bresu _ 0507 ( legI ) Bresu _ 3265 . Molecular masses are indicated in kDa by blue lines . Empty carets indicate position of modified ( glycosylated ) flagellin , whereas filled carets mark unmodified flagellin . Asterisk indicates a modification of flagellin by FlmGBs that does not require Pse or Leg . Immunoblots are representative of at least two independent experiments .

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

(B) Anti-FljKCc immunoblot analyses of whole cell lysates (cells) from C. crescentus (C.c.) mutant cultures expressing fljKBssyn (codon optimized for E. coli) and flmGBs from the replicative pMT463 plasmid, in the presence or absence of a compatible integrative plasmid carrying a legBs synthetic operon (pXGFP4-legBs) and in the presence or absence of an additional compatible replicative plasmid carrying Bresu_3267 (pMT375). In the presence of pXGFP4-legBs (integrated at the xylX locus) and pMT375-Bresu_3267, FljKBssyn migration is shifted toward higher molecular mass, indicative of glycosylation. Note that in the C.c. Δfljx6 background, the six flagellin-encoding genes have been deleted and the protein detected by the antibodies only corresponds to FljKBssyn. The legBs synthetic operon (in red) is composed of Bresu_3266, Bresu_0765, Bresu_0506, Bresu_3264, Bresu_0507 (legI) and Bresu_3265. Molecular masses are indicated in kDa by the blue lines. Empty carets indicate the position of modified (glycosylated) flagellin, whereas filled carets mark unmodified flagellin. Asterisk indicates a modification of flagellin by FlmGBs that does not require Pse or Leg. Immunoblots are representative of at least two independent experiments.


( C ) Anti - FljKCc immunoblot analyses of whole lysates from C . crescentus mutant cultures expressing FlmGCc , FlmGBs FlmGCc - Bschim chimera from replicative pSRK - Gm plasmid ( Plac ) in presence of a compatible integrative plasmid carrying a legBs synthetic operon ( pXGFP4 - legBs ) in presence or absence of an additional compatible replicative plasmid carrying Bresu _ 3267 ( pMT375 ) . Molecular masses are indicated in kDa by blue lines . Empty carets indicate position of modified ( glycosylated ) flagellin , whereas filled carets mark unmodified flagellin . Immunoblots are representative of at least two independent experiments .

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

(C) Anti-FljKCc immunoblot analyses of whole cell lysates from C. crescentus mutant cultures expressing FlmGCc, FlmGBs and FlmGCc-Bschim chimera from the replicative pSRK-Gm plasmid (Plac) in the presence of a compatible integrative plasmid carrying a legBs synthetic operon (pXGFP4-legBs) and in the presence or absence of an additional compatible replicative plasmid carrying Bresu_3267 (pMT375). Molecular masses are indicated in kDa by the blue lines. Empty carets indicate the position of modified (glycosylated) flagellin, whereas filled carets mark unmodified flagellin. Immunoblots are representative of at least two independent experiments.


Strep - Tactin pull down of GIDAnt ( strep - tagged at Gid8 C - terminus ) probing binding of Gid1057 - 292 Gid4117 - 362 to visualized with Coomassie - stained SDS - PAGE . experiment was performed with WT C - terminal deletion ( ∆ C ) of substrate receptors ( ∆ 289 - 292 ( FEIA ) ∆ 359 - 362 ( FEFA ) for Gid10 Gid4 , respectively ) WT mutant ( mut , Gid5W606A / Y613A / Q649A ) GIDAnt .

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Strep-Tactin pull down of GIDAnt (strep-tagged at Gid8 C-terminus) probing binding of Gid1057-292 and Gid4117-362 to the complex visualized with Coomassie-stained SDS-PAGE. The experiment was performed with WT and C-terminal deletion (∆C) of the substrate receptors (∆289-292 (FEIA) and ∆359-362 (FEFA) for Gid10 and Gid4, respectively) and WT and mutant (mut, Gid5W606A/Y613A/Q649A) GIDAnt.


Lysates from yeast strains expressing endogenously tagged 3xFLAG - Gid4 or 3x - FLAG - Gid10 that were grown in SD complete at 30 ° C ( no stress ) , SE complete for 19 hours ( EtOH ) , SD complete for 1 hour following 19 hour ethanol treatment ( EtOH rec . ) , 42 ° C for 1 hour ( HS ) , SD complete supplemented with 0 . 5M NaCl for 1 hour ( OS ) , or SD - N for 1 hour ( - N ) were run on an SDS - PAGE gel immunoblotted with αFLAG ( two exposures from same gel are shown ) αPGK .

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

Lysates from yeast strains expressing endogenously tagged 3xFLAG-Gid4 or 3x-FLAG-Gid10 that were grown in SD complete at 30°C (no stress), SE complete for 19 hours (EtOH), SD complete for 1 hour following 19 hour ethanol treatment (EtOH rec.), 42°C for 1 hour (HS), SD complete supplemented with 0.5M NaCl for 1 hour (OS), or SD-N for 1 hour (-N) were run on an SDS-PAGE gel and immunoblotted with αFLAG (two exposures from the same gel are shown) and αPGK.


Lysates from yeast strains expressing endogenously tagged 3xFLAG - Gid4 or 3xFLAG - Gid10 that were grown at 42 ° C for one hour , then returned to 30 ° C for indicated timepoints were run on an SDS - PAGE gel immunoblotted with αFLAG ( two exposures from same gel are shown ) αPGK .

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Lysates from yeast strains expressing endogenously tagged 3xFLAG-Gid4 or 3xFLAG-Gid10 that were grown at 42°C for one hour, and then returned to 30°C for the indicated timepoints were run on an SDS-PAGE gel and immunoblotted with αFLAG (two exposures from the same gel are shown) and αPGK.


Lysates from wildtype ∆ Gid2 yeast strains expressing endogenously tagged 3xFLAG - Gid10 that were grown at 42 ° C for one hour , then returned to 30 ° C for indicated timepoints were run on an SDS - PAGE gel immunoblotted with αFLAG αPGK .

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Lysates from wildtype and ∆Gid2 yeast strains expressing endogenously tagged 3xFLAG-Gid10 that were grown at 42°C for one hour, and then returned to 30°C for the indicated timepoints were run on an SDS-PAGE gel and immunoblotted with αFLAG and αPGK.


Lysates from wildtype , Gid5W606A , Y613A , Q649A , Gid10 ∆ C strains expressing endogenously tagged 3xFLAG - Gid10 that were grown at 42 ° C for one hour , then returned to 30 ° C for indicated timepoints were run on an SDS - PAGE gel immunoblotted with αFLAG αPGK .

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Lysates from wildtype, Gid5W606A,Y613A,Q649A, and Gid10∆C strains expressing endogenously tagged 3xFLAG-Gid10 that were grown at 42°C for one hour, and then returned to 30°C for the indicated timepoints were run on an SDS-PAGE gel and immunoblotted with αFLAG and αPGK.


Lysates from wildtype , ∆ Gid4 , Gid4 ∆ C strains expressing endogenously tagged 3xFLAG - Gid10 that were grown at 42 ° C for one hour , then returned to 30 ° C for indicated timepoints were run on an SDS - PAGE gel immunoblotted with αFLAG αPGK .

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

Lysates from wildtype, ∆Gid4, and Gid4∆C strains expressing endogenously tagged 3xFLAG-Gid10 that were grown at 42°C for one hour, and then returned to 30°C for the indicated timepoints were run on an SDS-PAGE gel and immunoblotted with αFLAG and αPGK.


Wildtype Gid10 overexpressing yeast strains expressing endogenously tagged Gid4 were grown for 19 hours in YPE , transitioned to YPD for indicated timepoints . Lysates were run on an SDS - PAGE gel immunoblotted with αFLAG αPGK . Points represent mean , error bars represent standard deviation ( n > 3 biological replicates ) .

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Wildtype and Gid10 overexpressing yeast strains expressing endogenously tagged Gid4 were grown for 19 hours in YPE, and transitioned to YPD for the indicated timepoints. Lysates were run on an SDS-PAGE gel and immunoblotted with αFLAG and αPGK. Points represent mean, error bars represent standard deviation (n>3 biological replicates).


In vitro competition probing ability of N - terminally 2xStrep - tagged Gid4 ( 2xS - Gid4 ) to exchange with Gid10 in GIDSR10 . after Strep - Tactin pull - down were visualized by Coomassie - stained SDS - PAGE .

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In vitro competition assay probing the ability of N-terminally 2xStrep-tagged Gid4 (2xS-Gid4) to exchange with Gid10 in the GIDSR10 complex. Proteins after Strep-Tactin pull-down were visualized by Coomassie-stained SDS-PAGE.


Art2 - 3xFLAG was immunocaptured from yeast grown in YPD incubated with GidSR10 , GidSR4 , GidAnt , or Gid10 for indicated timepoints . Progress of reaction was followed by αFLAG immunoblot .

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Art2-3xFLAG was immunocaptured from yeast cells grown in YPD and incubated with GidSR10, GidSR4, GidAnt, or Gid10 for the indicated timepoints. Progress of the reaction was followed by αFLAG immunoblot.


Mdh2 - 3xFLAG was immunocaptured from yeast following growth in YPE for 19h incubated with GidSR10 , GidSR4 , GidAnt , or Gid4 for indicated timepoints . Progress of reaction was followed by αFLAG immunoblot .

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Mdh2-3xFLAG was immunocaptured from yeast cells following growth in YPE for 19h and incubated with GidSR10, GidSR4, GidAnt, or Gid4 for the indicated timepoints. Progress of the reaction was followed by αFLAG immunoblot.


Art2 - 3xFLAG or Art2P2S - 3xFLAG was immunocaptured from yeast grown in YPD incubated with GidSR10 , or GidSR10 ∆ C for indicated timepoints . Progress of reaction was followed by αFLAG immunoblot .

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Art2-3xFLAG or Art2P2S-3xFLAG was immunocaptured from yeast cells grown in YPD and incubated with GidSR10, or GidSR10∆C for the indicated timepoints. Progress of the reaction was followed by αFLAG immunoblot.


In vitro ubiquitination probing ability of Gid10 Gid4 to promote ubiquitination of Art22 - 29 N - terminus its P2S mutant . Progress of reaction was monitored by scan of gel visualizing Art2 peptide with FAM appended to its C - terminus ( pep * ) .

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

In vitro ubiquitination assay probing the ability of Gid10 and Gid4 to promote ubiquitination of Art22-29 N-terminus and its P2S mutant. Progress of the reaction was monitored by fluorescent scan of the gel visualizing the Art2 peptide with FAM appended to its C-terminus (pep*).


A . Unified cycle map of unperturbed ( gray ) serum - starved ( 1d : blue , 7d : blue , N = 3007 ) . proliferative cycle ( dotted gray ) hypomitogenic arrest trajectory ( black dotted ) are indicated on map . Inset : Each treatment condition is shown individually on unified map ( other conditions are shown in lighter gray ) .

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

A. Unified cell cycle map of unperturbed (gray) and serum-starved cells (1d: light blue, 7d: dark blue, N = 3007 cells). The proliferative cell cycle (dotted gray line) and the hypomitogenic arrest trajectory (black dotted line) are indicated on the map. Inset: Each treatment condition is shown individually on the unified map (other conditions are shown in lighter gray).


( B ) Diffusion pseudotime , ( C ) p21 , ( D ) cyclin D1 , ( E ) content of unperturbed ( left panels ) or serum - starved ( right panels ) are plotted on map .

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

(B) Diffusion pseudotime, (C) p21, (D) cyclin D1, (E) DNA content of unperturbed (left panels) or serum-starved cells (right panels) are plotted on the map.


( F ) phospho / total RB of unperturbed ( left panels ) or serum - starved ( right panels ) are plotted on map .

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

(F) phospho/total RB of unperturbed (left panels) or serum-starved cells (right panels) are plotted on the map.


G . Time - lapse imaging of cyclinD1 - mVenus in unperturbed ( control , green ) serum - starved ( blue ) . were serum - starved for at least 8h prior to imaging . solid represents population median shaded indicates 95 % confidence interval . N = 105 control N = 111 starved .

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

G. Time-lapse imaging of cyclinD1-mVenus intensity in unperturbed (control, green) and serum-starved cells (blue). Cells were serum-starved for at least 8h prior to imaging. The solid line represents the population median and the shaded area indicates the 95% confidence interval. N = 105 control cells and N = 111 starved cells.


I . Median p27 abundance in serum - starved is plotted on map .

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I. Median nuclear p27 abundance in serum-starved cells is plotted on the map.


Unified cycle map of unperturbed ( gray ) etoposide - treated ( 1 μM ; 1d : green , 2d : green , 3d : green , 4d : darker green - see inset , N = 4315 ) . unperturbed cycle trajectory ( dotted gray ) two arrest trajectories ( into G02C G04C ; black dotted lines ) are indicated on map . Inset : Each condition is shown individually on map ( other conditions are shown in lighter gray ) .

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

Unified cell cycle map of unperturbed (gray) and etoposide-treated cells (1 μM; 1d: light green, 2d: green, 3d: dark green, 4d: darker green - see inset, N = 4315 cells). The unperturbed cell cycle trajectory (dotted gray line) and two arrest trajectories (into G02C and G04C; black dotted lines) are indicated on the map. Inset: Each condition is shown individually on the map (other conditions are shown in lighter gray).


B - E . ( B ) Diffusion pseudotime , ( C ) content , ( D ) phospho / total RB ( E ) p21 of unperturbed ( left panels ) or etoposide - treated ( right panels ) are plotted on arrest architecture . Median values are shown .

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

B-E. (B) Diffusion pseudotime, (C) DNA content, (D) phospho/total RB and (E) p21 of unperturbed (left panels) or etoposide-treated cells (right panels) are plotted on the arrest architecture. Median nuclear values are shown.