Figure 2B ) Representative images of metaphases from YFP : CENP - A ( green ) HeLa cells treated with Control ( top ) or H2A . Z . 2 ( bottom ) siRNA . The white arrowheads indicate the mis - aligned chromosomes . Scale bar : 10 μm . C ) Representative images of metaphase spreads from Control ( top ) or H2A . Z . 2 ( bottom ) siRNA - transfected HeLa cells after FISH with a Chr17 centromeric probe ( green ) . The arrowheads indicate the separated sister chromatids . Scale bar : 10 μm . D ) Representative images of HeLa cells transfected with Control ( top ) or H2A . Z . 2 ( bottom ) siRNA , fixed , and stained for Sgo1 ( green ) . Scale bar : 10 μm . E ) Quantification of Sgo1 localisation in pro - metaphase cells from the experiment in ( D ) . Error bar represents SD of two biological replicates . 35 pro - metaphase cells were analysed . J ) Representative images of HeLa YFP : CENP - A ( green ) mitotic cells after Control ( top ) or H2A . Z . 2 ( bottom ) siRNA treatment . Scale bar : 5 μm . K ) Violin plot of centromeric CENP - A intensity of pro - metaphase / metaphase cells from the experiments in J . ( Control si N = 197 H2A . Z . 2 si N = 260 ) . The bar represents the median . Data sets were statistically analysed using the Wilcoxon rank test in R . * * = p < 0 . 01 . L ) Representative images of HeLa mitotic cells stained for CENP - C after Control ( top ) or H2A . Z . 2 ( bottom ) siRNA treatment . Scale bar : 5 μm . M ) Violin plot of centromeric CENP - C intensity of pro - metaphase / metaphase cells from the experiments in L . ( Control si N = 1083 H2A . Z . 2 si N = 686 , from 3 biological replicas ) . The bars represent the median . Data sets were statistically analysed using the Wilcoxon rank test in R . * * * = p < 0 . 0001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2B) Representative images of metaphases from YFP:CENP-A (green) HeLa cells treated with Control (top) or H2A.Z.2 (bottom) siRNA. The white arrowheads indicate the mis-aligned chromosomes. Scale bar: 10 μm. C) Representative images of metaphase spreads from Control (top) or H2A.Z.2 (bottom) siRNA-transfected HeLa cells after FISH with a Chr17 centromeric probe (green). The arrowheads indicate the separated sister chromatids. Scale bar: 10 μm. D) Representative images of HeLa cells transfected with Control (top) or H2A.Z.2 (bottom) siRNA, fixed, and stained for Sgo1 (green). Scale bar: 10 μm. E) Quantification of Sgo1 localisation in pro-metaphase cells from the experiment in (D). Error bar represents SD of two biological replicates. 35 pro-metaphase cells were analysed. J) Representative images of HeLa YFP:CENP-A (green) mitotic cells after Control (top) or H2A.Z.2 (bottom) siRNA treatment. Scale bar: 5 μm. K) Violin plot of centromeric CENP-A intensity of pro-metaphase/metaphase cells from the experiments in J. (Control si N=197 H2A.Z.2 si N=260). The bar represents the median. Data sets were statistically analysed using the Wilcoxon rank test in R. **=p<0.01. L) Representative images of HeLa mitotic cells stained for CENP-C after Control (top) or H2A.Z.2 (bottom) siRNA treatment. Scale bar: 5 μm. M) Violin plot of centromeric CENP-C intensity of pro-metaphase/metaphase cells from the experiments in L. (Control si N=1083 H2A.Z.2 si N=686, from 3 biological replicas). The bars represent the median. Data sets were statistically analysed using the Wilcoxon rank test in R. ***=p<0.0001.


Figure 3D ) IGV analyses of H2A . Z localisation ( from ENCODE ) on Aurora B ( green ) and c - MYC ( blue ) genes showing H2A . Z enrichment at the TSS and at the upstream region respectively ( bottom panels ) . IGV analyses of ATAC seq peaks for Control si and H2A . Z . 1 si for Aurora B and MYC genes . The number in red represents the log2Ratio H2AZ . 1 si / Control si .

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Figure 3D) IGV analyses of H2A.Z localisation (from ENCODE) on Aurora B (green) and c-MYC (blue) genes showing H2A.Z enrichment at the TSS and at the upstream region respectively (bottom panels). IGV analyses of ATAC seq peaks for Control si and H2A.Z.1 si for Aurora B and MYC genes. The number in red represents the log2Ratio H2AZ.1 si/Control si.


Figure 4E ) Representative images of Hela cell stained for Ki67 . The arrows indicate the different staining patterns quantified : strong signal ( grey arrow ) , weak signal ( light brown arrow ) and no signal ( dark brown arrow ( scale bar 10μm ) . F ) Quantification of the experiment in ( E ) . Error bars indicate the standard deviation from 3 biological replicates Data sets were statistically analysed using a Chi square test . * * * = p < 0 . 001 . G ) Representative images of HeLa cells transfected with control ( top ) or H2A . Z . 1 ( bottom ) siRNA and stained for Lamin A / C ( green ) . Scale bar = 10μm .

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Figure 4E) Representative images of Hela cell stained for Ki67. The arrows indicate the different staining patterns quantified: strong signal (grey arrow), weak signal (light brown arrow) and no signal (dark brown arrow (scale bar 10μm). F) Quantification of the experiment in (E). Error bars indicate the standard deviation from 3 biological replicates Data sets were statistically analysed using a Chi square test. ***=p<0.001. G) Representative images of HeLa cells transfected with control (top) or H2A.Z.1 (bottom) siRNA and stained for Lamin A/C (green). Scale bar = 10μm.


Figure 5C ) Kaplan Meier survival curve for neuroblastoma patients with low ( red ) or high ( blue ) expression of H2AFZ ( H2A . Z . 1 ) obtained from R2 genomics .

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Figure 5C) Kaplan Meier survival curve for neuroblastoma patients with low (red) or high (blue) expression of H2AFZ (H2A.Z.1) obtained from R2 genomics.


Figure 1Immunofluorescent detection of Nanog and Esrrb protein in wild - type E14Tg2a ESCs cultured for three days in GMEMβ / FCS / LIF . ( A ) Widefield images of a representative colony . : Immunofluorescent detection of Nanog and Esrrb protein in wild - type E14Tg2a ESCs cultured for three days in GMEMβ / FCS / LIF . Quantification of the mean Nanog and Esrrb fluorescence levels measured in nuclei identified by automatic segmentation of single optical plane images obtained by confocal microscopy . Values are expressed in Arbitrary Units ( AU ) . Oct4 negative cells , also identified by immunostaining , were excluded from the analysis . The red line represents the moving average of the data distribution . Representative of three independent experiments each including at least 1000 nuclei . C : Colonies of TNG E - tdT ESCs showing GFP expression from Nanog and Esrrb - tdTomato fusion protein expression from Esrrb after three days in GMEMβ / FCS / LIF . D : Flow cytometric analysis of Esrrb and Nanog fluorescent reporter expression in SSEA1 + TNG E - tdT ESCs cultured for three days in GMEMβ / FCS / LIF with or without puromycin . E : Immunofluorescent detection and quantification of total Nanog and Esrrb protein in E14Tg2a ESCs overexpressing Nanog ( left ) or Esrrb ( right ) cultured for three days in GMEMβ / FCS / LIF . Parental E14Tg2as are shown as a reference . The red lines indicate the negative thresholds defined by staining ESCs differentiatied for 3 days in the absence of LIF and presence of retinoic acid . F : Flow cytometric analysis of Esrrb - GFP and Nanog - mCherry fusion protein expression in SSEA1 + NER ESCs cultured for three days in GMEMβ / FCS / LIF . G : Histograms showing expression levels of Esrrb - GFP and Nanog - mCherry from the respective targeted endogenous alleles in SSEA1 + NER ESCs overexpressing Nanog ( left ) or Esrrb ( right ) cultured for three days in GMEMβ / FCS / LIF . Parental NER ESCs are shown as a reference , and wild - type E14Tg2a set the negative thresholds . H : Comparative flow cytometric analysis of Esrrb - 2a - tdTomato and Nanog : GFP expression in undifferentiated SSEA - 1 + TNG E - 2a - tdT or Nanog - null ES ∆ N E - 2a - tdT ESCs cultured for three days in GMEMβ / FCS / LIF . Data are shown as dot plots ( top ) and histogram ( bottom ) .

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Figure 1Immunofluorescent detection of Nanog and Esrrb protein in wild-type E14Tg2a ESCs cultured for three days in GMEMβ/FCS/LIF. (A) Widefield images of a representative colony.: Immunofluorescent detection of Nanog and Esrrb protein in wild-type E14Tg2a ESCs cultured for three days in GMEMβ/FCS/LIF. Quantification of the mean Nanog and Esrrb fluorescence levels measured in nuclei identified by automatic segmentation of single optical plane images obtained by confocal microscopy. Values are expressed in Arbitrary Units (AU). Oct4 negative cells, also identified by immunostaining, were excluded from the analysis. The red line represents the moving average of the data distribution. Representative of three independent experiments each including at least 1000 nuclei.C: Colonies of TNG E-tdT ESCs showing GFP expression from Nanog and Esrrb-tdTomato fusion protein expression from Esrrb after three days in GMEMβ/FCS/LIF.D: Flow cytometric analysis of Esrrb and Nanog fluorescent reporter expression in SSEA1+ TNG E-tdT ESCs cultured for three days in GMEMβ/FCS/LIF with or without puromycin.E: Immunofluorescent detection and quantification of total Nanog and Esrrb protein in E14Tg2a ESCs overexpressing Nanog (left) or Esrrb (right) cultured for three days in GMEMβ/FCS/LIF. Parental E14Tg2as are shown as a reference. The red lines indicate the negative thresholds defined by staining ESCs differentiatied for 3 days in the absence of LIF and presence of retinoic acid.F: Flow cytometric analysis of Esrrb-GFP and Nanog-mCherry fusion protein expression in SSEA1+ NER ESCs cultured for three days in GMEMβ/FCS/LIF.G: Histograms showing expression levels of Esrrb-GFP and Nanog-mCherry from the respective targeted endogenous alleles in SSEA1+ NER ESCs overexpressing Nanog (left) or Esrrb (right) cultured for three days in GMEMβ/FCS/LIF. Parental NER ESCs are shown as a reference, and wild-type E14Tg2a set the negative thresholds.H: Comparative flow cytometric analysis of Esrrb-2a-tdTomato and Nanog:GFP expression in undifferentiated SSEA-1+ TNG E-2a-tdT or Nanog-null ES∆N E-2a-tdT ESCs cultured for three days in GMEMβ/FCS/LIF. Data are shown as dot plots (top) and histogram (bottom).


Figure 2A : SSEA - 1 + TNG E - tdT ESCs were cultured for three days in GMEMβ / FCS / LIF and sorted into GFP + / tdThigh ( Nanoghigh / Esrrbhigh ) or GFP - / tdTlow fractions ( Nanoglow / Esrrblow ) . The purity of the sorted populations ( > 99 . 5 % ) is shown ( day 0 ) . After replating in GMEMβ / FCS / LIF , SSEA - 1 + cells were analysed daily for Esrrb - tdTomato and Nanog : GFP expression . B , C : Immunofluorescent detection of Nanog and Esrrb protein in wild - type E14Tg2a ESCs cultured in N2B27 / LIF / 2i and released in GMEMβ / FCS / LIF for 1 , 2 or 3 days . Quantification of the mean Nanog and Esrrb fluorescence levels measured in nuclei identified by automatic segmentation of single optical plane images obtained by confocal microscopy . Oct4 negative cells , also identified by immunostaining , were excluded from the analysis . ( B ) The red vertical and horizontal lines indicate the negative thresholds defined by staining ESCs differentiatied for 3 days in the absence of LIF and presence of retinoic acid . The red trendline represents the moving average of the data distribution at day 3 . Representative of two independent experiments each including at least 1000 nuclei . ( C ) Histogram plots of the data presented in B . : Sorted SSEA - 1 + and Esrrb - GFPhigh / Nanog - mCherryhigh NER ESCs were replated in GMEMβ / FCS / LIF and SSEA - 1 + cells were analysed daily for Esrrb - GFP and Nanog - mCherry expression . Wild - type E14Tg2a set the negative thresholds . E : SSEA - 1 + TNG E - tdT ESCs from the sorted populations used above ( panel A , day 0 ) were replated at clonal density in GMEMβ / LIF / FCS or N2B27 / LIF / 2i and stained for alkaline phosphatase activity after 6 days . Error bars : standard deviation of the number of colonies observed in two independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A: SSEA-1+ TNG E-tdT ESCs were cultured for three days in GMEMβ/FCS/LIF and sorted into GFP+/tdThigh (Nanoghigh/Esrrbhigh) or GFP-/tdTlow fractions (Nanoglow/Esrrblow). The purity of the sorted populations (>99.5%) is shown (day 0). After replating in GMEMβ/FCS/LIF, SSEA-1+ cells were analysed daily for Esrrb-tdTomato and Nanog:GFP expression.B,C: Immunofluorescent detection of Nanog and Esrrb protein in wild-type E14Tg2a ESCs cultured in N2B27/LIF/2i and released in GMEMβ/FCS/LIF for 1, 2 or 3 days. Quantification of the mean Nanog and Esrrb fluorescence levels measured in nuclei identified by automatic segmentation of single optical plane images obtained by confocal microscopy. Oct4 negative cells, also identified by immunostaining, were excluded from the analysis. (B) The red vertical and horizontal lines indicate the negative thresholds defined by staining ESCs differentiatied for 3 days in the absence of LIF and presence of retinoic acid. The red trendline represents the moving average of the data distribution at day 3. Representative of two independent experiments each including at least 1000 nuclei. (C) Histogram plots of the data presented in B.: Sorted SSEA-1+ and Esrrb-GFPhigh/Nanog-mCherryhigh NER ESCs were replated in GMEMβ/FCS/LIF and SSEA-1+ cells were analysed daily for Esrrb-GFP and Nanog-mCherry expression. Wild-type E14Tg2a set the negative thresholds.E: SSEA-1+ TNG E-tdT ESCs from the sorted populations used above (panel A, day 0) were replated at clonal density in GMEMβ/LIF/FCS or N2B27/LIF/2i and stained for alkaline phosphatase activity after 6 days. Error bars: standard deviation of the number of colonies observed in two independent experiments.


Figure 3A : Flow cytometric analysis of GFP expression in SSEA - 1 + E - GFPd1 ESCs cultured in N2B27 / LIF / 2i or GMEMβ / LIF / FCS compared to wild - type E14Tg2a ESCs . B : SSEA - 1 + E - GFPd1 ESCs sorted into EsrrbHi , EsrrbMed or EsrrbNeg subpopulations . Purity of the sorted populations , assessed by flow cytometry , is indicated above the sorting gates . C : Quantitative Esrrb mRNA expression analysis of populations sorted Error bars : standard deviation of gene expression values measured in four independent experiments . D : Populations sorted were replated at clonal density in GMEMβ / LIF / FCS ( top ) or N2B27 / LIF / 2i ( bottom ) and scored for alkaline phosphatase after 6d . Error bars : standard deviation of the number of colonies observed in two independent experiments . E : Quantitative mRNA expression analysis of populations sorted Error bars : standard deviation of gene expression values measured in four independent experiments . F : Microarray analysis of populations sorted represented as a heatmap ordered by expression change between EsrrbHi and EsrrbNeg . Expression levels were measured from three independent experiments and all genes expressed in at least one sample are shown ( n = 14 , 553 ) . The central line plot summarises the average fold change per cell population compared to EsrrbHi cells ( log2 scale ) with the colour coding indicated below the heat map . Previously identified Nanog and Esrrb targets are indicated on the right . G : Pearson correlation coefficients of the gene expression profiles of EsrrbH ( H ) , EsrrbM ( M ) , Esrrb - ( Neg ) E - GFPd1 ESCs and embryo derived ( late bud stage ) TNG E - 2a - tdT EpiSCs ( EpiSC ) compared to those of naïve ESC and EpiLCH : Pearson correlation coefficients in gene expression obtained comparing EsrrbHi ( H ) , EsrrbMed ( M ) , EsrrbNeg ( Neg ) E - GFPd1 ESCs and embryo derived ( late bud stage ) TNG E - 2a - tdT EpiSCs ( EpiSC ) to epiblast / ectoderm cells from embryos dissected at successive developmental stages ( CAV , cavity ; PS , prestreak ; ES , early - streak ; MS , midstreak ; LMS , late midstreak ; LS , late streak ; OB / EB , no bud / early bud ; LB , late bud ) . This analysis was restricted to the top 500 differentially expressed gene identified at successive developmental stages sorting by Hotelling T²

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Figure 3A: Flow cytometric analysis of GFP expression in SSEA-1+ E-GFPd1 ESCs cultured in N2B27/LIF/2i or GMEMβ/LIF/FCS compared to wild-type E14Tg2a ESCs.B: SSEA-1+ E-GFPd1 ESCs sorted into EsrrbHi, EsrrbMed or EsrrbNeg subpopulations. Purity of the sorted populations, assessed by flow cytometry, is indicated above the sorting gates.C: Quantitative Esrrb mRNA expression analysis of populations sorted Error bars: standard deviation of gene expression values measured in four independent experiments.D: Populations sorted were replated at clonal density in GMEMβ/LIF/FCS (top) or N2B27/LIF/2i (bottom) and scored for alkaline phosphatase after 6d. Error bars: standard deviation of the number of colonies observed in two independent experiments.E: Quantitative mRNA expression analysis of populations sorted Error bars: standard deviation of gene expression values measured in four independent experiments.F: Microarray analysis of populations sorted represented as a heatmap ordered by expression change between EsrrbHi and EsrrbNeg. Expression levels were measured from three independent experiments and all genes expressed in at least one sample are shown (n = 14,553). The central line plot summarises the average fold change per cell population compared to EsrrbHi cells (log2 scale) with the colour coding indicated below the heat map. Previously identified Nanog and Esrrb targets are indicated on the right.G: Pearson correlation coefficients of the gene expression profiles of EsrrbH (H), EsrrbM (M), Esrrb- (Neg) E-GFPd1 ESCs and embryo derived (late bud stage) TNG E-2a-tdT EpiSCs (EpiSC) compared to those of naïve ESC and EpiLCH: Pearson correlation coefficients in gene expression obtained comparing EsrrbHi (H), EsrrbMed (M), EsrrbNeg (Neg) E-GFPd1 ESCs and embryo derived (late bud stage) TNG E-2a-tdT EpiSCs (EpiSC) to epiblast/ectoderm cells from embryos dissected at successive developmental stages (CAV, cavity; PS, prestreak; ES, early-streak; MS, midstreak; LMS, late midstreak; LS, late streak; OB/EB, no bud/early bud; LB, late bud). This analysis was restricted to the top 500 differentially expressed gene identified at successive developmental stages sorting by Hotelling T² score


Figure 4A : Percentage of methylated CpG dinucleotides profiled across the Esrrb and Nanog enhancer and the Nanog promoter in sorted SSEA - 1 + / EsrrbHi , EsrrbMed or EsrrbNeg E - GFPd1 ESCs and TNG E - 2a - tdT EpiSC . CpG methylation was assessed by measuring protection from digestion of the HpaII , AciI , Hin6I or TaqI restriction sites ( indicated by vertical lines in the gene structure maps derived from the mouse reference genome ( mm9 ) and expressed relative to the TSS in ESCs Values represent total CpG methylation levels ( 5mC + 5hmC ) . Error bars : standard deviation of the measures in four independent experiments . B : Methylated CpG dinucleotides across the Esrrb enhancer were assessed SSEA - 1 + / EsrrbHi E - GFPd1 ESCs were sorted and placed back in culture overnight before resorting and methylation analysis relative to Esrrb - E - GFPd1 ESCs . Error bars : standard deviation of the measures in three independent experiments . C : The sorted E - GFPd1 ESCs populations were assessed by immunoblot for Esrrb and Nanog ( left ) or Oct4 ( right ) by fluorescence - based detection ( Licor ) . D : Quantitative ChIP - PCR analysis of OCT4 , NANOG and ESRRB binding and histone modifications at the Esrrb and Nanog enhancers and the Nanog promoter in the sorted populations Error bars : standard deviation of the measures in two ( NANOG , ESRRB , H3K27ac ) or three ( OCT4 , H3K4me3 , H3K4me1 ) independent ChIP experiments , each performed on pooled chromatin from at least three independently sorted samples . The diagrams at the bottom show the approximate position of the regions analysed for transcription factor binding or histone modifications ( in color or black ) , along with the relative control genomic locations ( grey ) .

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Figure 4A: Percentage of methylated CpG dinucleotides profiled across the Esrrb and Nanog enhancer and the Nanog promoter in sorted SSEA-1+ / EsrrbHi, EsrrbMed or EsrrbNeg E-GFPd1 ESCs and TNG E-2a-tdT EpiSC. CpG methylation was assessed by measuring protection from digestion of the HpaII, AciI, Hin6I or TaqI restriction sites (indicated by vertical lines in the gene structure maps derived from the mouse reference genome (mm9) and expressed relative to the TSS in ESCs Values represent total CpG methylation levels (5mC + 5hmC). Error bars: standard deviation of the measures in four independent experiments.B: Methylated CpG dinucleotides across the Esrrb enhancer were assessed SSEA-1+ / EsrrbHi E-GFPd1 ESCs were sorted and placed back in culture overnight before resorting and methylation analysis relative to Esrrb- E-GFPd1 ESCs. Error bars: standard deviation of the measures in three independent experiments.C: The sorted E-GFPd1 ESCs populations were assessed by immunoblot for Esrrb and Nanog (left) or Oct4 (right) by fluorescence-based detection (Licor).D: Quantitative ChIP-PCR analysis of OCT4, NANOG and ESRRB binding and histone modifications at the Esrrb and Nanog enhancers and the Nanog promoter in the sorted populations Error bars: standard deviation of the measures in two (NANOG, ESRRB, H3K27ac) or three (OCT4, H3K4me3, H3K4me1) independent ChIP experiments, each performed on pooled chromatin from at least three independently sorted samples. The diagrams at the bottom show the approximate position of the regions analysed for transcription factor binding or histone modifications (in color or black), along with the relative control genomic locations (grey).


Figure 5Average binding profile of ( B ) OCT4 in binding peaks where binding in EsrrbNeg ESCs was kept , lost or gained relative to EsrrbHi E - GFPd1 ESCs . Read counts per base pair were normalised by library size and by subtracting the background ( IgG ) signal . EsrrbHi ( EH ) , EsrrbMed ( EM ) and EsrrbNeg ( EN ) Average binding profile of ( D ) NANOG in binding peaks where binding in EsrrbNeg ESCs was kept , lost or gained relative to EsrrbHi E - GFPd1 ESCs . Read counts per base pair were normalised by library size and by subtracting the background ( IgG ) signal . EsrrbHi ( EH ) , EsrrbMed ( EM ) and EsrrbNeg ( EN ) G : Boxplots showing the fold change in expression levels of genes proximal to Class I elements , Class II elements or both Class I and Class II elements in EsrrbHi ( EH ) , EsrrbMed ( EM ) or EsrrbNeg ( EN ) E - GFPd1 ESCs and EpiSC ( Epi ) . Boxes span the inter - quartile range ( IQR ) from the first to the third quartile . The line indicates the median . Whiskers extend up to 1 . 5 IQR , and outliers are plotted . H , I : Binding profiles of OCT4 and NANOG in the proximity of two genes representative of ( H ) Class I ( Tet2 ) or ( I ) Class II ( Sall1 ) in EsrrbHi , EsrrbMed or EsrrbNeg E - GFPd1 ESCs . The relative occupancies of NANOG and OCT4 at Tet2 or Sall1 ( positions highlighted by hatched boxes ) alongside expression levels of Tet2 and Sall1 mRNAs in EsrrbHi ( EH ) , EsrrbMed ( EM ) or EsrrbNeg ( EN ) GFPd1 ESCs and EpiSC ( Epi ) is shown at the bottom . Oct4 binding data from

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Average binding profile of (B) OCT4 in binding peaks where binding in EsrrbNeg ESCs was kept, lost or gained relative to EsrrbHi E-GFPd1 ESCs. Read counts per base pair were normalised by library size and by subtracting the background (IgG) signal. EsrrbHi (EH), EsrrbMed (EM) and EsrrbNeg (EN)Average binding profile of (D) NANOG in binding peaks where binding in EsrrbNeg ESCs was kept, lost or gained relative to EsrrbHi E-GFPd1 ESCs. Read counts per base pair were normalised by library size and by subtracting the background (IgG) signal. EsrrbHi (EH), EsrrbMed (EM) and EsrrbNeg (EN)G: Boxplots showing the fold change in expression levels of genes proximal to Class I elements, Class II elements or both Class I and Class II elements in EsrrbHi (EH), EsrrbMed (EM) or EsrrbNeg (EN) E-GFPd1 ESCs and EpiSC (Epi). Boxes span the inter-quartile range (IQR) from the first to the third quartile. The line indicates the median. Whiskers extend up to 1.5 IQR, and outliers are plotted.H, I: Binding profiles of OCT4 and NANOG in the proximity of two genes representative of (H) Class I (Tet2) or (I) Class II (Sall1) in EsrrbHi, EsrrbMed or EsrrbNeg E-GFPd1 ESCs. The relative occupancies of NANOG and OCT4 at Tet2 or Sall1 (positions highlighted by hatched boxes) alongside expression levels of Tet2 and Sall1 mRNAs in EsrrbHi (EH), EsrrbMed (EM) or EsrrbNeg (EN) GFPd1 ESCs and EpiSC (Epi) is shown at the bottom. Oct4 binding data from EpiSC


Figure 6 : Average binding profile of ESRRB , KLF4 , NANOG , OCT4 and SOX2 to Class I ( green ) , Class II ( orange ) or all Nanog and Oct4 bound ( grey dashed line ) elements in ESCs . Read counts per base pair were normalised by library size and by subtracting the background ( IgG ) signal . B : Average FAIRE profile or ChIP - Seq profiles for H3K27ac , OCT4 , Ep300 or OTX2 in ESCs or EpiLC for Class I and Class II elements .

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Figure 6: Average binding profile of ESRRB, KLF4, NANOG, OCT4 and SOX2 to Class I (green), Class II (orange) or all Nanog and Oct4 bound (grey dashed line) elements in ESCs. Read counts per base pair were normalised by library size and by subtracting the background (IgG) signal.B: Average FAIRE profile or ChIP-Seq profiles for H3K27ac, OCT4, Ep300 or OTX2 in ESCs or EpiLC for Class I and Class II elements.


Figure 1DU145 cells were transfected with the indicated siRNA ( control , siCTRL ; UBTD1 : siUBTD1pool or siUBTD1single1 or single2 ) for 24 h and plated on fibronectin - coated glass coverslips . ( A ) The scatter plot shows the apparent Young ' s moduli obtained by AFM analysis . Bars represent the median values of the distributions . The immunoblot shows UBTD1 siRNA knock - down efficiency . DU145 cells were transfected with the indicated siRNA ( control , siCTRL ; UBTD1 : siUBTD1pool or siUBTD1single1 or single2 ) for 24 h and plated on fibronectin - coated glass coverslips . ( B ) Immunoblots ( left ) and quantification ( right ) show levels of GST - ROCK associated GTP - bound RhoA ( RhoA - GTP ) and total RhoA in lysates ( RhoA ) in confluent cells . Immunoblot of UBTD1 shows the level of siRNA depletion . DU145 cells were transfected with the indicated siRNA ( control , siCTRL ; UBTD1 : siUBTD1pool or siUBTD1single1 or single2 ) for 24 h and plated on fibronectin - coated glass coverslips . ( C ) Representative confocal images of focal adhesion ( paxillin ) and actin cytoskeleton ( phalloidin ) and outlines of focal adhesions using threshold - based image . Immunoblot of UBTD1 shows the level of siRNA - mediated depletion . ( D ) Cell area , number and area of focal adhesion were quantified ( ImageJ ) using the analysis as shown ( C ) . DU145 cells were transfected with the indicated siRNA ( control , siCTRL ; UBTD1 : siUBTD1pool or siUBTD1single1 or single2 ) for 24 h and plated on fibronectin - coated glass coverslips . Sub - confluent DU145 ( E ) cells were seeded on two matrices of different stiffnesses ( 4 and 12 kPa ) . Representative heat map ( left ) and quantification ( right ) showing contractile forces generate by cells plated on 4kPa or 12kPa hydrogel . A549 cells were transfected with the indicated siRNA ( control , siCTRL ; UBTD1 : siUBTD1pool or siUBTD1single1 or single2 ) for 24 h and plated on fibronectin - coated glass coverslips . Sub - confluent A549 ( F ) cells were seeded on two matrices of different stiffnesses ( 4 and 12 kPa ) . Representative heat map ( left ) and quantification ( right ) showing contractile forces generate by cells plated on 4kPa or 12kPa hydrogel . DU145 cells were transfected with the indicated siRNA ( control , siCTRL ; UBTD1 : siUBTD1pool or siUBTD1single1 or single2 ) for 24 h and plated on fibronectin - coated glass coverslips . ( G ) Confluent DU145 cells were seeded on 12 kPa matrix . Representative heat map ( left ) and quantification ( right ) showing contractile forces generate by cells plated on 12kPa hydrogel . DU145 cells were transfected with the indicated siRNA ( control , siCTRL ; UBTD1 : siUBTD1pool or siUBTD1single1 or single2 ) for 24 h and plated on fibronectin - coated glass coverslips . ( H ) Representative confocal images of YAP and UBTD1 localization on two matrices of different stiffness . DU145 cells were grown on collagen / acrylamide - coated glass coverslips ( soft ) or directly on glass coverslips ( stiff ) . Nuclei were stained with DAPI ( Blue ) on the MERGE image .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool or siUBTD1single1 or single2) for 24 h and plated on fibronectin-coated glass coverslips. (A) The scatter plot shows the apparent Young's moduli obtained by AFM analysis. Bars represent the median values of the distributions. The immunoblot shows UBTD1 siRNA knock-down efficiency.DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool or siUBTD1single1 or single2) for 24 h and plated on fibronectin-coated glass coverslips. (B) Immunoblots (left) and quantification (right) show levels of GST-ROCK associated GTP-bound RhoA (RhoA-GTP) and total RhoA in lysates (RhoA) in confluent cells. Immunoblot of UBTD1 shows the level of siRNA depletion.DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool or siUBTD1single1 or single2) for 24 h and plated on fibronectin-coated glass coverslips. (C) Representative confocal images of focal adhesion (paxillin) and actin cytoskeleton (phalloidin) and outlines of focal adhesions using threshold-based image. Immunoblot of UBTD1 shows the level of siRNA-mediated depletion. (D) Cell area, number and area of focal adhesion were quantified (ImageJ) using the analysis as shown (C).DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool or siUBTD1single1 or single2) for 24 h and plated on fibronectin-coated glass coverslips. Sub-confluent DU145 (E) cells were seeded on two matrices of different stiffnesses (4 and 12 kPa). Representative heat map (left) and quantification (right) showing contractile forces generate by cells plated on 4kPa or 12kPa hydrogel.A549 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool or siUBTD1single1 or single2) for 24 h and plated on fibronectin-coated glass coverslips. Sub-confluent A549 (F) cells were seeded on two matrices of different stiffnesses (4 and 12 kPa). Representative heat map (left) and quantification (right) showing contractile forces generate by cells plated on 4kPa or 12kPa hydrogel.DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool or siUBTD1single1 or single2) for 24 h and plated on fibronectin-coated glass coverslips. (G) Confluent DU145 cells were seeded on 12 kPa matrix. Representative heat map (left) and quantification (right) showing contractile forces generate by cells plated on 12kPa hydrogel.DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool or siUBTD1single1 or single2) for 24 h and plated on fibronectin-coated glass coverslips. (H) Representative confocal images of YAP and UBTD1 localization on two matrices of different stiffness. DU145 cells were grown on collagen/acrylamide-coated glass coverslips (soft) or directly on glass coverslips (stiff). Nuclei were stained with DAPI (Blue) on the MERGE image.


Figure 2 ( A ) Immunoblot of DU145 and A549 cells at various cell densities ranging from 20 % to 100 % confluence showing the increased expression of UBTD1 and β - catenin with cell confluency . E - cadherin was used as a positive control . Tubulin was used as a loading control . , immunofluorescent labelling of UBTD1 and E - cadherin ( B ) at the cell - cell contact in confluent DU145 cells and analyzed by confocal microscopy . Right , representative histogram of a co - localization profile prepared with RGB profiler plugin of ImageJ . immunofluorescent labelling of UBTD1 and β - catenin ( C ) at the cell - cell contact in confluent DU145 cells and analyzed by confocal microscopy . Right , representative histogram of a co - localization profile prepared with RGB profiler plugin of ImageJ . Proximal ligation assay monitoring ( left ) and quantification ( right ) of UBTD1 association with E - cadherin or β - catenin in DU145 cells transfected with the indicated siRNA ( control , siCTRL ; UBTD1 : siUBTD1pool ) . Nuclei were stained with DAPI ( Blue ) on the MERGE image . Immunoblots of UBTD1 , β - catenin and YAP after subcellular fractionation of confluent DU145 cells transfected with the indicated siRNA ( control , siCTRL ; UBTD1 : siUBTD1pool ) . EGFR , RhoGDI and Lamin A / C were used as controls for the membrane ( M ) , cytosol ( C ) and nucleus ( N ) compartments , respectively . Co - immunoprecipitation between endogenous β - catenin and UBTD1 was performed in confluent DU145 cells . IgG isotype was used as a negative control . ( G ) Representative confocal images of β - catenin and UBTD1 in DU145 under resting ( Ctrl ) , after calcium chelator treatment ( EGTA ) and recovery ( Rec ) conditions . Nuclei were stained with DAPI ( Blue ) on the MERGE image . ( H ) Representative confocal images of β - catenin and UBTD1 in DU145 after cell scattering induced by HGF treatment ( 24 h ) . Nuclei were stained with DAPI ( Blue ) on the MERGE image .

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Figure 2(A) Immunoblot of DU145 and A549 cells at various cell densities ranging from 20% to 100% confluence showing the increased expression of UBTD1 and β-catenin with cell confluency. E-cadherin was used as a positive control. Tubulin was used as a loading control., immunofluorescent labelling of UBTD1 and E-cadherin (B) at the cell-cell contact in confluent DU145 cells and analyzed by confocal microscopy. Right, representative histogram of a co-localization profile prepared with RGB profiler plugin of ImageJ.immunofluorescent labelling of UBTD1 and β-catenin (C) at the cell-cell contact in confluent DU145 cells and analyzed by confocal microscopy. Right, representative histogram of a co-localization profile prepared with RGB profiler plugin of ImageJ.Proximal ligation assay monitoring (left) and quantification (right) of UBTD1 association with E-cadherin or β-catenin in DU145 cells transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool). Nuclei were stained with DAPI (Blue) on the MERGE image.Immunoblots of UBTD1, β-catenin and YAP after subcellular fractionation of confluent DU145 cells transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool). EGFR, RhoGDI and Lamin A/C were used as controls for the membrane (M), cytosol (C) and nucleus (N) compartments, respectively.Co-immunoprecipitation between endogenous β-catenin and UBTD1 was performed in confluent DU145 cells. IgG isotype was used as a negative control.(G) Representative confocal images of β-catenin and UBTD1 in DU145 under resting (Ctrl), after calcium chelator treatment (EGTA) and recovery (Rec) conditions. Nuclei were stained with DAPI (Blue) on the MERGE image.(H) Representative confocal images of β-catenin and UBTD1 in DU145 after cell scattering induced by HGF treatment (24 h). Nuclei were stained with DAPI (Blue) on the MERGE image.


Figure 3Immunoblot of DU145 and A549 cells at various cell densities ranging from 20 % to 100 % confluence showing the increased expression of UBTD1 , p - YAP ( ser127 ) and YAP levels with the cell confluency . Tubulin was used as a loading control . DU145 cells were transfected for 48h with the indicated siRNA ( control , siCTRL ; UBTD1 , siUBTD1pool ) . ( B ) Immunoblots of p - YAP ( ser127 ) , YAP and UBTD1 ( left ) and quantification of YAP and p - YAP / YAP levels ( right ) in control or UBTD1 - depleted DU145 at low and high cell density . Immunoblot of UBTD1 shows the level of siRNA - mediated depletion . Actin was used as a loading controls . , immunoblots for the loading control ( actin ) and UBTD1 knock - down efficiency ( UBTD1 ) are duplicated because they belong to the same experiment shown in both panels . DU145 cells were transfected for 48h with the indicated siRNA ( control , siCTRL ; UBTD1 , siUBTD1pool ) . ( C ) Immunoblots of the Hippo signaling pathway using phosphorylated ( p - ser909LATS and p - thr183MST1 / p - thr180MST2 ) and total LATS , MST at low and high DU145 cells density . Immunoblot of UBTD1 shows the level of siRNA - mediated depletion . Actin and Rho - GDI were used as loading controls . , immunoblots for the loading control ( actin ) and UBTD1 knock - down efficiency ( UBTD1 ) are duplicated because they belong to the same experiment shown in both panels . DU145 cells were transfected for 48h with the indicated siRNA ( control , siCTRL ; UBTD1 , siUBTD1pool ) . Immunoblots ( up ) and quantification ( down ) of YAP levels in confluent ( D ) DU145 UBTD1 depleted cells treated or not with cycloheximide ( CHX ) for 2h . Immunoblot of UBTD1 shows the level of siRNA - mediated depletion . Actin was used as a loading control . A549 cells were transfected for 48h with the indicated siRNA ( control , siCTRL ; UBTD1 , siUBTD1pool ) . Immunoblots ( up ) and quantification ( down ) of YAP levels in confluent ( E ) A549 UBTD1 depleted cells treated or not with cycloheximide ( CHX ) for 2h . Immunoblot of UBTD1 shows the level of siRNA - mediated depletion . Actin was used as a loading control . DU145 cells or A549 cells were transfected for 48h with the indicated siRNA ( control , siCTRL ; UBTD1 , siUBTD1pool ) . Immunoblots ( up ) and quantification ( down ) showing YAP levels in UBTD1 depleted confluent DU145 A549 cells treated or not with MG132 for 6h . Immunoblot of UBTD1 shows the level of siRNA - mediated depletion . Actin was used as a loading control .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Immunoblot of DU145 and A549 cells at various cell densities ranging from 20% to 100% confluence showing the increased expression of UBTD1, p-YAP (ser127) and YAP levels with the cell confluency. Tubulin was used as a loading control.DU145 cells were transfected for 48h with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool). (B) Immunoblots of p-YAP (ser127), YAP and UBTD1 (left) and quantification of YAP and p-YAP/YAP levels (right) in control or UBTD1-depleted DU145 at low and high cell density. Immunoblot of UBTD1 shows the level of siRNA-mediated depletion. Actin was used as a loading controls. , immunoblots for the loading control (actin) and UBTD1 knock-down efficiency (UBTD1) are duplicated because they belong to the same experiment shown in both panels.DU145 cells were transfected for 48h with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool). (C) Immunoblots of the Hippo signaling pathway using phosphorylated (p-ser909LATS and p-thr183MST1/p-thr180MST2) and total LATS, MST at low and high DU145 cells density. Immunoblot of UBTD1 shows the level of siRNA-mediated depletion. Actin and Rho-GDI were used as loading controls. , immunoblots for the loading control (actin) and UBTD1 knock-down efficiency (UBTD1) are duplicated because they belong to the same experiment shown in both panels.DU145 cells were transfected for 48h with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool). Immunoblots (up) and quantification (down) of YAP levels in confluent (D) DU145 UBTD1 depleted cells treated or not with cycloheximide (CHX) for 2h. Immunoblot of UBTD1 shows the level of siRNA-mediated depletion. Actin was used as a loading control.A549 cells were transfected for 48h with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool). Immunoblots (up) and quantification (down) of YAP levels in confluent (E) A549 UBTD1 depleted cells treated or not with cycloheximide (CHX) for 2h. Immunoblot of UBTD1 shows the level of siRNA-mediated depletion. Actin was used as a loading control.DU145 cells or A549 cells were transfected for 48h with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool). Immunoblots (up) and quantification (down) showing YAP levels in UBTD1 depleted confluent DU145 A549 cells treated or not with MG132 for 6h. Immunoblot of UBTD1 shows the level of siRNA-mediated depletion. Actin was used as a loading control.


Figure 4 ( A ) Co - immunoprecipitation in DU145 and A549 cells between endogenous YAP and UBTD1 . YAP was used as bait . The IgG isotype was used as a negative control . ( B ) Co - immunoprecipitation in DU145 and A549 cells between endogenous YAP and UBTD1 . UBTD1 was used as bait . The IgG isotype was used as a negative control . ( C ) Proximal ligation assay monitoring ( left ) and quantification ( right ) of UBTD1 associated with YAP of DU145 and A549 cells transfected with the indicated siRNA ( control , siCTRL ; UBTD1 : siUBTD1pool ) . Nuclei were stained with DAPI ( Blue ) on the MERGE image . ( D ) Co - immunoprecipitation in confluent DU145 cells between endogenous β - TrCP , Ubch5c and UBTD1 . UBTD1 was used as bait . IgG isotype was used as a negative control . ( E ) Co - immunoprecipitation in confluent DU145 cells between endogenous β - TrCP , Ubch5c , YAP and β - catenin . DU145 cells were transfected with the indicated siRNA ( control , siCTRL ; UBTD1 , siUBTD1pool or siUBTD1single ) . β - TrCP was used as bait . IgG isotype was used as a negative control . Immunoblot of UBTD1 shows the level of siRNA - mediated depletion . Proximal ligation assay monitoring ( F ) of β - TrCP associated with Ubch5 , YAP or β - catenin in DU145 cells transfected with the indicated siRNA ( control , siCTRL ; UBTD1 : siUBTD1pool ) . Nuclei were stained with DAPI ( Blue ) on the MERGE image . quantification ( G ) of β - TrCP associated with Ubch5 , YAP or β - catenin in DU145 cells transfected with the indicated siRNA ( control , siCTRL ; UBTD1 : siUBTD1pool ) . ( H ) Immunoblots show YAP ubiquitylation in HEK cells in different experimental conditions . Cells were transfected , as indicated , with expression vectors of histidine - tagged ubiquitin ( His - Ub ) together with control siRNA or UBTD1 siRNA . His - Ub crosslinked forms of YAP were purified ( IP : His ) and the immunoblot of YAP showed YAP ubiquitylation . The immunoblot of YAP ( lower panel ) was performed in parallel to verify the amounts of YAP protein engaged in the His - Ub purifications . The immunoblot of UBTD1 shows the level of siRNA depletion .

O O B-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Co-immunoprecipitation in DU145 and A549 cells between endogenous YAP and UBTD1. YAP was used as bait. The IgG isotype was used as a negative control.(B) Co-immunoprecipitation in DU145 and A549 cells between endogenous YAP and UBTD1. UBTD1 was used as bait. The IgG isotype was used as a negative control.(C) Proximal ligation assay monitoring (left) and quantification (right) of UBTD1 associated with YAP of DU145 and A549 cells transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool). Nuclei were stained with DAPI (Blue) on the MERGE image.(D) Co-immunoprecipitation in confluent DU145 cells between endogenous β-TrCP, Ubch5c and UBTD1. UBTD1 was used as bait. IgG isotype was used as a negative control.(E) Co-immunoprecipitation in confluent DU145 cells between endogenous β-TrCP, Ubch5c, YAP and β-catenin. DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool or siUBTD1single). β-TrCP was used as bait. IgG isotype was used as a negative control. Immunoblot of UBTD1 shows the level of siRNA-mediated depletion.Proximal ligation assay monitoring (F) of β-TrCP associated with Ubch5, YAP or β-catenin in DU145 cells transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool). Nuclei were stained with DAPI (Blue) on the MERGE image.quantification (G) of β-TrCP associated with Ubch5, YAP or β-catenin in DU145 cells transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool).(H) Immunoblots show YAP ubiquitylation in HEK cells in different experimental conditions. Cells were transfected, as indicated, with expression vectors of histidine-tagged ubiquitin (His-Ub) together with control siRNA or UBTD1 siRNA. His-Ub crosslinked forms of YAP were purified (IP: His) and the immunoblot of YAP showed YAP ubiquitylation. The immunoblot of YAP (lower panel) was performed in parallel to verify the amounts of YAP protein engaged in the His-Ub purifications. The immunoblot of UBTD1 shows the level of siRNA depletion.


Figure 5DU145 cells were transfected with the indicated siRNA ( control , siCTRL ; UBTD1 , siUBTD1pool ) . ( A ) Representative wide - field immunofluorescence images ( left ) and quantification ( right ) showing endogenous YAP nuclear translocation in DU145 cells after UBTD1 depletion . Nuclei were stained with DAPI ( Blue ) on the MERGE image . DU145 cells were transfected with the indicated siRNA ( control , siCTRL ; UBTD1 , siUBTD1pool ) . ( B ) Quantification of CTGF1 , CYR61 and BIRC5 mRNA levels . ( C ) Immunoblots of YAP in DU145 cells depleted ( siUBTD1single2 ) or not ( siCTRL ) for UBTD1 and transfected or not with GFP - UBTD1 ( + ) . Immunoblot of UBTD1 shows the level of siRNA - mediated depletion . Immunoblot of GFP shows the level of UBTD1 transfection . CHC was used as a loading control . ( D ) Representative confocal immunofluorescence images ( left ) and quantification ( right ) showing endogenous YAP nuclear translocation in DU145 cells depleted ( siUBTD1single2 ) or not ( siCTRL ) for UBTD1 and transfected or not with GFP - UBTD1 . Nuclei were stained with DAPI ( Blue ) on the MERGE image . ( E ) Immunoblots of p - YAP and YAP in DU145 control cells ( siCTRL ) or depleted for LATS1 and LATS2 ( siLATS1 + siLATS2 ) and / or UBTD1 ( siUBTD1pool ) . Immunoblot of p - YAP reflects the activity of the Hippo pathway . Immunoblot of UBTD1 and LATS show siRNA knock - down efficiency . Actin was used as a loading control . Representative confocal immunofluorescence images ( F ) showing endogenous YAP nuclear translocation in DU145 control cells ( siCTRL ) or depleted for LATS1 and LATS2 ( siLATS1 + siLATS2 ) and / or UBTD1 ( siUBTD1pool ) . Nuclei were stained with DAPI ( Blue ) on the MERGE image . quantification ( G ) showing endogenous YAP nuclear translocation in DU145 control cells ( siCTRL ) or depleted for LATS1 and LATS2 ( siLATS1 + siLATS2 ) and / or UBTD1 ( siUBTD1pool ) . ( H ) Immunoblots of YAP in DU145 control ( siCTRL ) or depleted for ROCK1 ( siROCK1 ) or ROCK2 ( siROCK2 ) and / or UBTD1 ( siUBTD1pool ) . Immunoblots of UBTD1 , ROCK1 and ROCK2 show the level of siRNA knock - down efficiency . Actin was used as a loading control . Representative confocal immunofluorescence images ( I ) showing endogenous YAP localization in DU145 control cells ( siCTRL ) or depleted for ROCK1 ( siROCK1 ) or ROCK2 ( siROCK2 ) and / or UBTD1 ( siUBTD1pool ) . Nuclei were stained with DAPI ( Blue ) on the MERGE image . quantification ( J ) showing endogenous YAP localization in DU145 control cells ( siCTRL ) or depleted for ROCK1 ( siROCK1 ) or ROCK2 ( siROCK2 ) and / or UBTD1 ( siUBTD1pool ) . ( K ) Quantification of CTGF1 , CYR61 , UBTD1 and BIRC5 mRNA in DU145 control cells ( siCTRL ) or depleted for ROCK1 ( siROCK1 ) or ROCK2 ( siROCK2 ) and / or UBTD1 ( siUBTD1pool ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool). (A) Representative wide-field immunofluorescence images (left) and quantification (right) showing endogenous YAP nuclear translocation in DU145 cells after UBTD1 depletion. Nuclei were stained with DAPI (Blue) on the MERGE image.DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool). (B) Quantification of CTGF1, CYR61 and BIRC5 mRNA levels.(C) Immunoblots of YAP in DU145 cells depleted (siUBTD1single2) or not (siCTRL) for UBTD1 and transfected or not with GFP-UBTD1 (+). Immunoblot of UBTD1 shows the level of siRNA-mediated depletion. Immunoblot of GFP shows the level of UBTD1 transfection. CHC was used as a loading control.(D) Representative confocal immunofluorescence images (left) and quantification (right) showing endogenous YAP nuclear translocation in DU145 cells depleted (siUBTD1single2) or not (siCTRL) for UBTD1 and transfected or not with GFP-UBTD1. Nuclei were stained with DAPI (Blue) on the MERGE image.(E) Immunoblots of p-YAP and YAP in DU145 control cells (siCTRL) or depleted for LATS1 and LATS2 (siLATS1+siLATS2) and/or UBTD1 (siUBTD1pool). Immunoblot of p-YAP reflects the activity of the Hippo pathway. Immunoblot of UBTD1 and LATS show siRNA knock-down efficiency. Actin was used as a loading control.Representative confocal immunofluorescence images (F) showing endogenous YAP nuclear translocation in DU145 control cells (siCTRL) or depleted for LATS1 and LATS2 (siLATS1+siLATS2) and/or UBTD1 (siUBTD1pool). Nuclei were stained with DAPI (Blue) on the MERGE image.quantification (G) showing endogenous YAP nuclear translocation in DU145 control cells (siCTRL) or depleted for LATS1 and LATS2 (siLATS1+siLATS2) and/or UBTD1 (siUBTD1pool).(H) Immunoblots of YAP in DU145 control (siCTRL) or depleted for ROCK1 (siROCK1) or ROCK2 (siROCK2) and/or UBTD1 (siUBTD1pool). Immunoblots of UBTD1, ROCK1 and ROCK2 show the level of siRNA knock-down efficiency. Actin was used as a loading control.Representative confocal immunofluorescence images (I) showing endogenous YAP localization in DU145 control cells (siCTRL) or depleted for ROCK1 (siROCK1) or ROCK2 (siROCK2) and/or UBTD1 (siUBTD1pool). Nuclei were stained with DAPI (Blue) on the MERGE image.quantification (J) showing endogenous YAP localization in DU145 control cells (siCTRL) or depleted for ROCK1 (siROCK1) or ROCK2 (siROCK2) and/or UBTD1 (siUBTD1pool).(K) Quantification of CTGF1, CYR61, UBTD1 and BIRC5 mRNA in DU145 control cells (siCTRL) or depleted for ROCK1 (siROCK1) or ROCK2 (siROCK2) and/or UBTD1 (siUBTD1pool).


Figure 6DU145 cells were transfected with the indicated siRNA ( control , siCTRL ; UBTD1 , siUBTD1pool or siUBTD1single1 ; UBTD1 + YAP + TAZ , siUBTD1single1 + siYAPsingle1 + siTAZsingle1 or siUBTD1single1 + siYAPsingle2 + siTAZsingle2 ) . ( A ) Quantification of EMT marker genes ( vimentin , slug , snail , fibronectin , α - SMA , E - cadherin ) and UBTD1 . ( B ) Immunoblots of EMT markers ( vimentin , E - cadherin , slug ) . Immunoblot of UBTD1 shows siRNA knock - down efficiency . Hsp90 and tubulin were used as loading controls . DU145 cells were transfected with the indicated siRNA ( control , siCTRL ; UBTD1 , siUBTD1pool or siUBTD1single1 ; UBTD1 + YAP + TAZ , siUBTD1single1 + siYAPsingle1 + siTAZsingle1 or siUBTD1single1 + siYAPsingle2 + siTAZsingle2 ) . ( C ) Quantification of DU145 cell migration in Transwell chamber inserts . DU145 cells were transfected with the indicated siRNA ( control , siCTRL ; UBTD1 , siUBTD1pool or siUBTD1single1 ; UBTD1 + YAP + TAZ , siUBTD1single1 + siYAPsingle1 + siTAZsingle1 or siUBTD1single1 + siYAPsingle2 + siTAZsingle2 ) . ( D ) Quantification of DU145 cell invasion in Transwell chamber inserts . ( E ) Invasive properties of human prostate tumoroids depleted for UBTD1 . ( F ) Quantification of UBTD1 mRNA levels in prostate tumor ( n = 70 ) and adjacent normal tissues ( n = 47 ) . ( G ) Representative UBTD1 immunohistochemistry staining on sections of human prostate tumor and normal tissues ( n = 8 ) . ( H ) Representative UBTD1 immunohistochemistry staining on sections of human lung tumor and normal tissues ( n = 6 ) . ( I ) The levels of UBTD1 mRNA in grade 3 prostate adenocarcinoma patients ( 56 patients ) are correlated with overall survival ( OS ) . OS was calculated from patient subgroups with mRNA levels that were less or greater than the first quartile value . ( J ) The levels of UBTD1 mRNA in lung adenocarcinoma patients ( 522 patients ) are correlated with OS .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O B-PANEL_START O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O

Figure 6DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool or siUBTD1single1; UBTD1 +YAP+TAZ, siUBTD1single1+siYAPsingle1+ siTAZsingle1 or siUBTD1single1+siYAPsingle2+ siTAZsingle2). (A) Quantification of EMT marker genes (vimentin, slug, snail, fibronectin, α-SMA, E-cadherin) and UBTD1.(B) Immunoblots of EMT markers (vimentin, E-cadherin, slug). Immunoblot of UBTD1 shows siRNA knock-down efficiency. Hsp90 and tubulin were used as loading controls.DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool or siUBTD1single1; UBTD1 +YAP+TAZ, siUBTD1single1+siYAPsingle1+ siTAZsingle1 or siUBTD1single1+siYAPsingle2+ siTAZsingle2). (C) Quantification of DU145 cell migration in Transwell chamber inserts.DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool or siUBTD1single1; UBTD1 +YAP+TAZ, siUBTD1single1+siYAPsingle1+ siTAZsingle1 or siUBTD1single1+siYAPsingle2+ siTAZsingle2). (D) Quantification of DU145 cell invasion in Transwell chamber inserts.(E) Invasive properties of human prostate tumoroids depleted for UBTD1.(F) Quantification of UBTD1 mRNA levels in prostate tumor (n=70) and adjacent normal tissues (n=47).(G) Representative UBTD1 immunohistochemistry staining on sections of human prostate tumor and normal tissues (n=8).(H) Representative UBTD1 immunohistochemistry staining on sections of human lung tumor and normal tissues (n=6).(I) The levels of UBTD1 mRNA in grade 3 prostate adenocarcinoma patients (56 patients) are correlated with overall survival (OS). OS was calculated from patient subgroups with mRNA levels that were less or greater than the first quartile value.(J) The levels of UBTD1 mRNA in lung adenocarcinoma patients (522 patients) are correlated with OS.


Figure 1Example time trace when no binding event occurs ( green laser excitation ) . EmCCD image shows signal from green and red channels before the addition of Cy3 - labelled DNA when illuminated with green laser . Size of EmCCD image is 25µm x 50 µm . Example time trace showing a binding event ( green laser excitation ) . EmCCD image shows signal from green and red channels after the addition of Cy3 - labeled DNA when illuminated with green laser . Size of EmCCD image is 25µm x 50 µm .

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Figure 1Example time trace when no binding event occurs (green laser excitation). EmCCD image shows signal from green and red channels before the addition of Cy3-labelled DNA when illuminated with green laser. Size of EmCCD image is 25µm x 50 µm.   Example time trace showing a binding event (green laser excitation). EmCCD image shows signal from green and red channels after the addition of Cy3-labeled DNA when illuminated with green laser. Size of EmCCD image is 25µm x 50 µm.


Figure 2Example time traces when DNA containing 3 PAM sequences is added ( top ) and when DNA containing a single PAM is added showing both short and long binding events ( bottom ) . FRET histograms of all binding events for each construct . Y axis represents total number of counts , divided by the number of immobilized molecules in the field of view . Bar plot of all dwelltime values for all constructs showing short binding events for all DNA and long events only for the constructs containing PAM sequences . The times shown are mean values of dwelltimes obtained during four different experiments on four different days . Error bars represent standard error of the mean . Example dwelltime histograms from negative control and a construct containing a single PAM . Negative control dwelltime distribution is characterized by a single exponential decay ( top ) and dwelltime distribution for PAM - containing DNA is fitted by a double - exponential decay ( blue line ) ( bottom ) . Equation for the double exponential decay fit : y = A1e - t / τ1 + A2e - t / τ2 ; ΔA1 = 796 . 4 ΔA2 = 28 . 3 . Red line shows what a single - exponential decay fit for such distribution would be . Errors represent standard error of the mean . Scatter plot showing the average dwelltime ( Δτav ) values for the cases of 2 ( d2 ) and 4 ( d4 ) nucleotide separation between the PAM sites . The values are averages of four measurements over four different days for each case . Error bars represent the standard error of the mean . The average dwelltime was obtained using the equation τav = ( A1Δτ12 + A2τ22 ) / ( A1Δτ1 + A2Δτ2 ) Example trace showing direct FRET transitions for a 5 - PAM containing DNA construct with separation between PAM sites being 4 nucleotides . Histogram showing the percentage of events that are longer than τ1 showing FRET fluctuations for multiple PAM constructs with PAM separation of 4 nucleotides . The values are averages of four measurements over four different days . The error bars represent standard error of the mean .

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Figure 2Example time traces when DNA containing 3 PAM sequences is added (top) and when DNA containing a single PAM is added showing both short and long binding events (bottom).FRET histograms of all binding events for each construct. Y axis represents total number of counts, divided by the number of immobilized molecules in the field of view.Bar plot of all dwelltime values for all constructs showing short binding events for all DNA and long events only for the constructs containing PAM sequences. The times shown are mean values of dwelltimes obtained during four different experiments on four different days. Error bars represent standard error of the mean.Example dwelltime histograms from negative control and a construct containing a single PAM. Negative control dwelltime distribution is characterized by a single exponential decay (top) and dwelltime distribution for PAM-containing DNA is fitted by a double-exponential decay (blue line) (bottom). Equation for the double exponential decay fit: y=A1e-t/τ1 + A2e-t/τ2; ΔA1= 796.4 ΔA2= 28.3. Red line shows what a single-exponential decay fit for such distribution would be. Errors represent standard error of the mean.Scatter plot showing the average dwelltime (Δτav) values for the cases of 2 (d2) and 4 (d4) nucleotide separation between the PAM sites. The values are averages of four measurements over four different days for each case. Error bars represent the standard error of the mean. The average dwelltime was obtained using the equation τav=(A1Δτ12+A2τ22)/(A1Δτ1+A2Δτ2)Example trace showing direct FRET transitions for a 5-PAM containing DNA construct with separation between PAM sites being 4 nucleotides.Histogram showing the percentage of events that are longer than τ1 showing FRET fluctuations for multiple PAM constructs with PAM separation of 4 nucleotides. The values are averages of four measurements over four different days. The error bars represent standard error of the mean.


Figure 3FRET histogram of all binding events from the construct containing a single PAM next to the partial target . Y axis represents total number of counts . Example FRET traces from single - target multiple - PAM experiments . Top trace shows an example of a single - step binding event and a binding event that starts at a lower FRET state before transitioning to productive binding high - FRET state . Bottom trace shows an example of FRET fluctuations between the high - FRET state and lower FRET states . A histogram showing the percentage of dynamic . The percentage values were obtained from five experiments on five different days . Error bars represent standard error of the mean . The percentage of events showing dynamic binding behavior was calculated by dividing the number of binding events that showed such behavior by the total number of binding events . A scatter plot showing the time Cas9 spends in a lower FRET state before transitioning to a high - FRET state . The dwelltimes increase with the number of PAMs adjacent to the target . Such dwelltime could not be determined for a construct containing a single PAM . The values were obtained from five experiments on five different days . Error bars represent standard error of the mean .

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Figure 3FRET histogram of all binding events from the construct containing a single PAM next to the partial target. Y axis represents total number of counts.Example FRET traces from single-target multiple-PAM experiments. Top trace shows an example of a single-step binding event and a binding event that starts at a lower FRET state before transitioning to productive binding high-FRET state. Bottom trace shows an example of FRET fluctuations between the high-FRET state and lower FRET states.A histogram showing the percentage of dynamic. The percentage values were obtained from five experiments on five different days. Error bars represent standard error of the mean. The percentage of events showing dynamic binding behavior was calculated by dividing the number of binding events that showed such behavior by the total number of binding events.A scatter plot showing the time Cas9 spends in a lower FRET state before transitioning to a high-FRET state. The dwelltimes increase with the number of PAMs adjacent to the target. Such dwelltime could not be determined for a construct containing a single PAM. The values were obtained from five experiments on five different days. Error bars represent standard error of the mean.


Figure 4Example fluorescence traces showing Cas9 transitioning between H and L targets with increasing target distances ( right ) . FRET histograms constructed from events that show fluctuations showing two FRET peaks corresponding to binding to either target . High FRET peak stays the same in each histogram , but lower FRET peak moves to lower FRET values as the distance between targets increases . A scatter plot showing the probability of Cas9 transitioning between targets before dissociation . The values are averages from five measurements and error bars represent standard error of the mean . The transition probability ( p = T / ( T + D ) ) was calculated by summing up all the number of FRET transitions ( T ) and dividing T by the sum of T and the total number of dissociation events ( D ) . A histogram showing all dwelltimes from single - target controls and tandem - target experiments . τ1 is similar for single - target control and tandem target experiments . τ2 only appears in tandem target experiments and is over 20 - fold higher than τ1 or τst . The values represent the mean dwelltimes that were obtained by fitting dwelltime distributions by either single or double exponential decay function from five measurements . Error bars represent standard error of the mean .

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Figure 4Example fluorescence traces showing Cas9 transitioning between H and L targets with increasing target distances (right). FRET histograms constructed from events that show fluctuations showing two FRET peaks corresponding to binding to either target. High FRET peak stays the same in each histogram, but lower FRET peak moves to lower FRET values as the distance between targets increases.A scatter plot showing the probability of Cas9 transitioning between targets before dissociation. The values are averages from five measurements and error bars represent standard error of the mean. The transition probability (p = T / (T+D)) was calculated by summing up all the number of FRET transitions (T) and dividing T by the sum of T and the total number of dissociation events (D).A histogram showing all dwelltimes from single-target controls and tandem-target experiments. τ1 is similar for single-target control and tandem target experiments. τ2 only appears in tandem target experiments and is over 20-fold higher than τ1 or τst. The values represent the mean dwelltimes that were obtained by fitting dwelltime distributions by either single or double exponential decay function from five measurements. Error bars represent standard error of the mean.


Figure 5Histogram showing the percentage of binding events starting at either target . The values are averages of five measurements over three different days . Error bars represent standard error of the mean . An example trace showing the addition of Cy3 - labeled DNA target and the first binding event . Δτon is indicated . Scatter plot showing the on - target binding rate ( kon - target ) for each construct . Binding rate decreases moderately with increasing number of adjacent PAMs . The values are averages of four measurements over three days . Error bars represent standard error of the mean . kon - target was calculated using the equation kon - target = 1 / ( CxΔτon ) where C is the molarity of DNA target in the channel .

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Figure 5Histogram showing the percentage of binding events starting at either target. The values are averages of five measurements over three different days. Error bars represent standard error of the mean.An example trace showing the addition of Cy3-labeled DNA target and the first binding event. Δτon is indicated.Scatter plot showing the on-target binding rate (kon-target) for each construct. Binding rate decreases moderately with increasing number of adjacent PAMs. The values are averages of four measurements over three days. Error bars represent standard error of the mean. kon-target was calculated using the equation kon-target=1/ (CxΔτon) where C is the molarity of DNA target in the channel.


Figure 1E . Microscale thermophoresis binding experiment for PlexA1 - 4 - mVenus and Sema1becto ( red ) or Sema1becto - mutA ( blue ) or Sema1becto - mutB ( green , ) or Sema1becto - mutA + B ( orange ) . The Sema1becto wild - type binding results are as reported previously ( Rozbesky et al . , 2019 ) . All data were collected at the same time , error bars represent s . d . of three technical replicates .

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Figure 1E. Microscale thermophoresis binding experiment for PlexA1-4-mVenus and Sema1becto (red) or Sema1becto-mutA (blue) or Sema1becto-mutB (green,) or Sema1becto-mutA+B (orange). The Sema1becto wild-type binding results are as reported previously (Rozbesky et al., 2019). All data were collected at the same time, error bars represent s.d. of three technical replicates.


Figure 2A . FRET - FLIM in live COS - 7 cells shows cell surface PlexA - Sema1b cis interaction . Sema1b mutants reveal that both head - to - head and side - on orientations of the PlexA - Sema1b complex are involved in the cis interaction . The cis interaction between Sema1a and PlexA is not observed . The boxcharts represent the average lifetime . Box limits indicate the 25th and 75th percentiles , centred lines show the median , squares represent sample means , whiskers extend 1 . 5 - fold the interquartile range from the 25th and 75th percentiles , p - value was calculated by one - way analysis of variance ( ANOVA ) . * P < 0 . 05 , * * * P < 0 . 001B . Representative negative stain class averages of PlexAecto correlate well with the ring - like conformation of mouse PlxnA1 . The ring - like conformation of mouse PlxnA1 is shown in surface representation rainbow colour ramped from blue ( N terminus ) to red ( C terminus ) . Scale bar , 10 nm . E . In vitro FRET - FLIM measurement of the PlexA - FRET Biosensor shows that the PlexA ectodomain undergoes a conformational change upon treatment with Sema1b resulting in more open conformation . The boxcharts represent the average lifetime . Box limits indicate the 25th and 75th percentiles , centred lines show the median , squares represent sample means , whiskers extend 1 . 5 - fold the interquartile range from the 25th and 75th percentiles , p - value was calculated by one - way analysis of variance ( ANOVA ) . * * * P < 0 . 001 . F . Relative change of COS - 7 cells surface area upon treatment with Sema1a - Fc . Cells expressing PlexAFL - mClover or PlexAFL - mCover and Sema1b - mRuby2 wild - type or mutants were treated with purified Sema1a - Fc at a final concentration of 5 . 8 µM . Images were acquired every minute for 30 minutes . Cell surface area was calculated using ImageJ before and after stimulation . Data are presented as means ± sem . * P < 0 . 05 , * * * P < 0 . 001 , p - value calculated by one - way analysis of variance ( ANOVA ) .

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Figure 2A. FRET-FLIM in live COS-7 cells shows cell surface PlexA-Sema1b cis interaction. Sema1b mutants reveal that both head-to-head and side-on orientations of the PlexA-Sema1b complex are involved in the cis interaction. The cis interaction between Sema1a and PlexA is not observed. The boxcharts represent the average lifetime. Box limits indicate the 25th and 75th percentiles, centred lines show the median, squares represent sample means, whiskers extend 1.5-fold the interquartile range from the 25th and 75th percentiles, p-value was calculated by one-way analysis of variance (ANOVA). *P<0.05, ***P<0.001B. Representative negative stain class averages of PlexAecto correlate well with the ring-like conformation of mouse PlxnA1. The ring-like conformation of mouse PlxnA1 is shown in surface representation rainbow colour ramped from blue (N terminus) to red (C terminus). Scale bar, 10 nm.E. In vitro FRET-FLIM measurement of the PlexA-FRET Biosensor shows that the PlexA ectodomain undergoes a conformational change upon treatment with Sema1b resulting in more open conformation. The boxcharts represent the average lifetime. Box limits indicate the 25th and 75th percentiles, centred lines show the median, squares represent sample means, whiskers extend 1.5-fold the interquartile range from the 25th and 75th percentiles, p-value was calculated by one-way analysis of variance (ANOVA). ***P<0.001.F. Relative change of COS-7 cells surface area upon treatment with Sema1a-Fc. Cells expressing PlexAFL-mClover or PlexAFL-mCover and Sema1b-mRuby2 wild-type or mutants were treated with purified Sema1a-Fc at a final concentration of 5.8 µM. Images were acquired every minute for 30 minutes. Cell surface area was calculated using ImageJ before and after stimulation. Data are presented as means ± sem. *P<0.05, ***P<0.001, p-value calculated by one-way analysis of variance (ANOVA).


Figure 3A . Cultured dorsal root ganglion ( DRG ) neurons from E12 . 5 Sema6A knockout ( KO ) embryos were transfected with EGFP , EGFP - tagged wild - type ( WT ) Sema6A ( Sema6A - WT ) or different EGFP - tagged Sema6A mutants ( Sema6A - mutA , Sema6A - mutB , and Sema6A - mutA + B ) at 1 day in vitro ( DIV1 ) and treated with purified Sema6A - Fc at a concentration of 75 nM for 1 hour at 37 ° C at DIV4 . Transfection of EGFP was used as a control ( full collapse ) . DRG neurons from Sema6A KO embryos acquired sensitivity to Sema6A and growth cone collapse was observed in three independent experiments . Transfection of Sema6A - WT , Sema6A - mutA and Sema6A - mutB but not of EGFP or Sema6A - mutantA + B prevented Sema6A - mediated growth cone collapse ( Fisher ' s exact test p < 0 . 0001 ) . Quantification of growth cone collapse was performed using a growth cone morphology matrix ( lower part panel A ) . Data is presented as percentage of morphologically distinct growth cones , divided ( represented by the dotted line ) in two categories , from uncollapsed ( 1 - 4 ) to fully collapsed ( 5 - 8 ) . Totally , 20 - 40 growth cones were analyzed for each condition per experiment ( n = 3 experiments ) . Scale bar 20 µm . B . Representative images of growth cones of mouse DRG neurons transfected with WT or mutant constructs treated with 75 nM Sema6A - Fc and visualized with immunofluorescent staining to detect GFP ( green ) and phalloidin ( red ) . Average intensity images for all different constructs are shown in grey color . Scale bar 20 µm .

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Figure 3A. Cultured dorsal root ganglion (DRG) neurons from E12.5 Sema6A knockout (KO) embryos were transfected with EGFP, EGFP-tagged wild-type (WT) Sema6A (Sema6A-WT) or different EGFP-tagged Sema6A mutants (Sema6A-mutA, Sema6A-mutB, and Sema6A-mutA+B) at 1 day in vitro (DIV1) and treated with purified Sema6A-Fc at a concentration of 75 nM for 1 hour at 37°C at DIV4. Transfection of EGFP was used as a control (full collapse). DRG neurons from Sema6A KO embryos acquired sensitivity to Sema6A and growth cone collapse was observed in three independent experiments. Transfection of Sema6A-WT, Sema6A-mutA and Sema6A-mutB but not of EGFP or Sema6A-mutantA+B prevented Sema6A-mediated growth cone collapse (Fisher's exact test p<0.0001). Quantification of growth cone collapse was performed using a growth cone morphology matrix (lower part panel A). Data is presented as percentage of morphologically distinct growth cones, divided (represented by the dotted line) in two categories, from uncollapsed (1-4) to fully collapsed (5-8). Totally, 20-40 growth cones were analyzed for each condition per experiment (n=3 experiments). Scale bar 20 µm.B. Representative images of growth cones of mouse DRG neurons transfected with WT or mutant constructs treated with 75 nM Sema6A-Fc and visualized with immunofluorescent staining to detect GFP (green) and phalloidin (red). Average intensity images for all different constructs are shown in grey color. Scale bar 20 µm.


Figure 1A . Representative H & E staining and immunohistochemical analysis of the androgen receptor ( AR ) in the dorsolateral prostate ( DLP ) of PtenL2 / L2 and Pten ( i ) pe - / - mice , sham - operated ( sham ) or castrated ( CTX ) at 3 months after gene inactivation ( AGI ) , and analyzed at 1 month after surgery . Scale bars : 100 µm . N = 3 mice per condition . F . Violin plots depicting transcript levels of androgen - responsive genes in luminal - C cells of sham and CTX Pten ( i ) pe - / - mice . P - values were determined by Wilcoxon rank sum test . H . Violin plots depicting the transcript levels of inflammation - related genes in luminal - C cells of sham and CTX Pten ( i ) pe - / - mice . P - values were determined by Wilcoxon rank sum test . J , K . Violin plots depicting the transcript levels of hypoxia - related genes ( J ) and plasticity - related genes ( K ) in luminal - C cells of sham and CTX Pten ( i ) pe - / - mice . P - values were determined by Wilcoxon rank sum test .

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Figure 1A. Representative H&E staining and immunohistochemical analysis of the androgen receptor (AR) in the dorsolateral prostate (DLP) of PtenL2/L2 and Pten(i)pe-/- mice, sham-operated (sham) or castrated (CTX) at 3 months after gene inactivation (AGI), and analyzed at 1 month after surgery. Scale bars: 100 µm. N=3 mice per condition.F. Violin plots depicting transcript levels of androgen-responsive genes in luminal-C cells of sham and CTX Pten(i)pe-/- mice. P-values were determined by Wilcoxon rank sum test.H. Violin plots depicting the transcript levels of inflammation-related genes in luminal-C cells of sham and CTX Pten(i)pe-/- mice. P-values were determined by Wilcoxon rank sum test.J, K. Violin plots depicting the transcript levels of hypoxia-related genes (J) and plasticity-related genes (K) in luminal-C cells of sham and CTX Pten(i)pe-/- mice. P-values were determined by Wilcoxon rank sum test.


Figure 2C . Gland areas in DLP of sham and CTX control and Pten / Hif1a ( i ) pe - / - mice . Five glands per mouse were quantified . N = 3 mice / condition . G . Representative immunohistochemical detection of cleaved caspase 3 ( CC3 ) in DLP of Pten ( i ) pe - / - and Pten / Hif1a ( i ) pe - / - mice , sham or CTX at 3 months AGI , and analyzed 1 or 1 , 2 , 3 and 30 days after surgery , respectively . N = 3 mice / condition . I . Western blot analysis of CC3 in protein lysates of organoids generated from prostates of Pten ( i ) pe - / - and Pten / Hif1a ( i ) pe - / - mice at 3 months AGI , and treated with enzalutamide ( Enz ) for 24 hours at the indicated concentrations . β - actin was used as a loading control .

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Figure 2C. Gland areas in DLP of sham and CTX control and Pten/Hif1a(i)pe-/- mice. Five glands per mouse were quantified. N=3 mice/condition.G. Representative immunohistochemical detection of cleaved caspase 3 (CC3) in DLP of Pten(i)pe-/- and Pten/Hif1a(i)pe-/- mice, sham or CTX at 3 months AGI, and analyzed 1 or 1, 2, 3 and 30 days after surgery, respectively. N=3 mice/condition.I. Western blot analysis of CC3 in protein lysates of organoids generated from prostates of Pten(i)pe-/- and Pten/Hif1a(i)pe-/- mice at 3 months AGI, and treated with enzalutamide (Enz) for 24 hours at the indicated concentrations. β-actin was used as a loading control.


Figure 3B . Representative H & E staining of DLP of Pten / Hif1a ( i ) pe - / - mice sham and CTX at 3 months AGI and analyzed at 5 months after surgery . N = 4 - 5 mice / condition . C . Gland areas in DLP of control and Pten / Hif1a ( i ) pe - / - mice , sham and CTX at 3 months AGI and analyzed at 5 months after surgery . N = 3 - 4 mice / condition . Five glands per mouse were quantified . Data presented are mean , and error bars correspond to SEM . E . Representative immunohistochemical detection of AR and TROP2 in DLP of Pten / Hif1a ( i ) pe - / - mice , sham and CTX at 3 months AGI and analyzed at 5 months after surgery . N = 3 - 4 mice / condition .

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Figure 3B. Representative H&E staining of DLP of Pten/Hif1a(i)pe-/- mice sham and CTX at 3 months AGI and analyzed at 5 months after surgery. N=4-5 mice/condition. C. Gland areas in DLP of control and Pten/Hif1a(i)pe-/- mice, sham and CTX at 3 months AGI and analyzed at 5 months after surgery. N=3-4 mice/condition. Five glands per mouse were quantified. Data presented are mean, and error bars correspond to SEM.E. Representative immunohistochemical detection of AR and TROP2 in DLP of Pten/Hif1a(i)pe-/- mice, sham and CTX at 3 months AGI and analyzed at 5 months after surgery. N = 3-4 mice/condition.


Figure 4D . Representative western blot analysis of CPARP and CC3 in human C4 - 2B PCa cells treated for 24 h with PX - 478 , increasing concentrations of enzalutamide ( Enz ) , or a combination of both . Beta - actin was used as loading control . F . Representative H & E staining of DLP of sham and CTX Pten ( i ) pe - / - mice , treated with vehicle or PX - 478 , as depicted in ( E ) . Scale bar : 100 µm . N = 3 - 4 mice / condition . H . Gland areas of DLP of sham and CTX Pten ( i ) pe - / - mice , treated with vehicle or PX - 478 , as depicted in ( E ) . N = 3 - 4 mice / condition . Data presented are mean ± SEM . Five glands per mouse were quantified ,

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Figure 4D. Representative western blot analysis of CPARP and CC3 in human C4-2B PCa cells treated for 24 h with PX-478, increasing concentrations of enzalutamide (Enz), or a combination of both. Beta-actin was used as loading control.F. Representative H&E staining of DLP of sham and CTX Pten(i)pe-/- mice, treated with vehicle or PX-478, as depicted in (E). Scale bar: 100 µm. N=3-4 mice/condition.H. Gland areas of DLP of sham and CTX Pten(i)pe-/- mice, treated with vehicle or PX-478, as depicted in (E). N=3-4 mice/condition. Data presented are mean ± SEM. Five glands per mouse were quantified,