Relative enrichment of indicated proteins in MTA pulldowns from wild type ( WT ) or Mbd3 - null ( Mbd3KO ) ES cells , normalised to 2x MTA proteins . NuRD components comprising the remodelling subunit are labelled in blue , those comprising the deacetylase subunit in red . As MTA proteins are the bait in these experiments , they may be isolated more efficiently than their interaction partners . Error bars represent standard deviation from three ( Mta1 and Mta2 ) or six ( Mta3 ) replicate pulldowns .

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Relative enrichment of indicated proteins in MTA pulldowns from wild type (WT) or Mbd3-null (Mbd3KO) ES cells, normalised to 2x MTA proteins. NuRD components comprising the remodelling subunit are labelled in blue, those comprising the deacetylase subunit in red. As MTA proteins are the bait in these experiments, they may be isolated more efficiently than their interaction partners. Error bars represent standard deviation from three (Mta1 and Mta2) or six (Mta3) replicate pulldowns.


ChIP - seq peaks identified for Mta1 and Mta2 ( left ) , Mta1 and Mta3 ( middle ) or Mta2 and Mta3 ( right ) are plotted by enrichment for each protein . Peaks called for both proteins are indicated in the overlap .

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ChIP-seq peaks identified for Mta1 and Mta2 (left), Mta1 and Mta3 (middle) or Mta2 and Mta3 (right) are plotted by enrichment for each protein. Peaks called for both proteins are indicated in the overlap.


ChIP - seq enrichment for indicated proteins or histone modifications is plotted across different subsets of Mta - bound sites . Mta1 / 2 / 3 refers to peaks identified in all three ChIP - seq datasets , whereas " Mta1 Only " , " Mta2 Only " or " Mta3 Only " refer to peaks only called for that protein .

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ChIP-seq enrichment for indicated proteins or histone modifications is plotted across different subsets of Mta-bound sites. Mta1/2/3 refers to peaks identified in all three ChIP-seq datasets, whereas "Mta1 Only", "Mta2 Only" or "Mta3 Only" refer to peaks only called for that protein.


Average enrichment of density plots are plotted for different subsets of Mta ChIP - seq peaks .

Average enrichment of density plots are plotted for different subsets of Mta ChIP-seq peaks.


Western blots of wild type ( Control ) , double or triple - null ES cell nuclear extract was probed with antibodies indicated at right . LaminB1 acts as a loading control . Approximate sizes are indicated in kDa .

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Western blots of wild type (Control), double or triple-null ES cell nuclear extract was probed with antibodies indicated at right. LaminB1 acts as a loading control. Approximate sizes are indicated in kDa.


Phase - contrast images of wild type or Mta123 ∆ ES cells in 2i + LIF conditions . Scale bars represent

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Phase-contrast images of wild type or Mta123∆ ES cells in 2i+LIF conditions. Scale bars represent 100µ


Western blots of anti - Chd4 immunoprecipitation of nuclear extract from indicated cell lines ( top ) probed with antibodies indicated at right . For the Gatad2b and Mbd3 blots , arrowheads indicate target proteins , while an asterisk marks a non - specific IgG bands . 10 % of the input was loaded into " Input " lanes . Approximate sizes are indicated in kDa .

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Western blots of anti-Chd4 immunoprecipitation of nuclear extract from indicated cell lines (top) probed with antibodies indicated at right. For the Gatad2b and Mbd3 blots, arrowheads indicate target proteins, while an asterisk marks a non-specific IgG bands. 10% of the input was loaded into "Input" lanes. Approximate sizes are indicated in kDa.


Western blot of a time course of Mta3 deletion in Mta1 ∆ Mta2 ∆ Mta3Flox / Flox : Cre - ER ( Mta12 ∆ 3F / F ) or Control ( Mta12 ∆ 3F / F without Cre - ER ) ES cells probed for indicated NuRD component proteins or RNA Polymerase II as a loading control ( αRNAPII ) . The time course is indicated at the top as Days + tamoxifen .

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Western blot of a time course of Mta3 deletion in Mta1∆Mta2∆Mta3Flox/Flox: Cre-ER (Mta12∆3F/F) or Control (Mta12∆3F/F without Cre-ER) ES cells probed for indicated NuRD component proteins or RNA Polymerase II as a loading control (αRNAPII). The time course is indicated at the top as Days + tamoxifen.


Western blot showing rescue of Mta123 ∆ ES cells by ectopic expression of Mta1 , Mta2 or Mta3 from a transgene ( TG ) . Total RNA Polymerase II acts as a loading control ( αRNAPII ) . For all western blots molecular weight is indicated at left in kDa .

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Western blot showing rescue of Mta123∆ ES cells by ectopic expression of Mta1, Mta2 or Mta3 from a transgene (TG). Total RNA Polymerase II acts as a loading control (αRNAPII). For all western blots molecular weight is indicated at left in kDa.


Comparison of gene expression in Mta123 ∆ ES cells and wild type ( WT ) ES cells . Each circle represents a gene : red indicates spike - in controls , blue indicates genes that are not differentially expressed to a significant degree , and green indicates differentially expressed genes ( 2404 increased , 1293 decreased ) defined with an adjusted p - value < 0 . 05 and a log2 fold - change > 1 . N = 3 for each genotype .

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

Comparison of gene expression in Mta123∆ ES cells and wild type (WT) ES cells. Each circle represents a gene: red indicates spike-in controls, blue indicates genes that are not differentially expressed to a significant degree, and green indicates differentially expressed genes (2404 increased, 1293 decreased) defined with an adjusted p-value < 0.05 and a log2 fold-change > 1. N=3 for each genotype.


Fold change in gene expression is plotted for different subsets of genes . All genes are plotted in black , while subsets of genes located nearest to ChIP - seq peaks for the indicated proteins + Chd4 and which show significant changes in expression compared to wild type cells are plotted in dashed coloured lines as indicated . The number of genes in each of the Mta categories are : all genes ( 32271 ) , all differentially expressed ( n = 3701 ) , Mta1 + 2 + 3 ( n = 1738 ) , Mta1 + 2 ( n = 1460 ) , Mta1 ( n = 1020 ) and Mta2 ( n = 924 ) .

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Fold change in gene expression is plotted for different subsets of genes. All genes are plotted in black, while subsets of genes located nearest to ChIP-seq peaks for the indicated proteins +Chd4 and which show significant changes in expression compared to wild type cells are plotted in dashed coloured lines as indicated. The number of genes in each of the Mta categories are: all genes (32271), all differentially expressed (n=3701), Mta1+2+3 (n=1738), Mta1+2 (n=1460), Mta1 (n=1020) and Mta2 (n=924).


Principal component analysis of RNAseq data from ES cell lines of indicated genotypes in either self - renewing conditions ( 2i ) or after 48 hours in the absence of two inhibitors and LIF ( Diff48 ) . Each point represents a biological replicate .

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Principal component analysis of RNAseq data from ES cell lines of indicated genotypes in either self-renewing conditions (2i) or after 48 hours in the absence of two inhibitors and LIF (Diff48). Each point represents a biological replicate.


Genes associated with indicated GO terms plotted by fold change in expression in Mta123 ∆ ES cells ( x - axis ) or Mbd3 ∆ ES cells ( y - axis ) . Genes are coloured if they are differentially expressed ( log2 fold - change > 1 and adjusted p - value < 0 . 05 ) in either comparison as indicated . The dotted lines show the fold - change cut - off of 2 . GO - terms were identified using David v . 6 . 8 using a Benjamini score with a cutoff of 0 . 05 .

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Genes associated with indicated GO terms plotted by fold change in expression in Mta123∆ ES cells (x-axis) or Mbd3∆ ES cells (y-axis). Genes are coloured if they are differentially expressed (log2 fold-change > 1 and adjusted p-value < 0.05) in either comparison as indicated. The dotted lines show the fold-change cut-off of 2. GO-terms were identified using David v.6.8 using a Benjamini score with a cutoff of 0.05.


Genes associated with core NuRD peaks , with an MTA - only peak , with a Chd4 peak and an MTA - peak but not an Mbd3 peak ( Chd4 + Mta - Mbd3 genes ) , or with an MTA - peak but not a Chd4 peak ( MTA - only genes ) were divided into quartiles based upon expression levels in wild type cells . Change in gene expression is plotted on the x - axis and calculated significance of the expression change on the y - axis . Those genes significantly misexpressed ( | log2FC | > 1 ; p ≤ 0 . 05 ) are indicated in red , genes showing no significant change are plotted in black . The percentage of genes within each quartile which is significantly misexpressed is indicated at the top of each plot .

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

Genes associated with core NuRD peaks, with an MTA-only peak, with a Chd4 peak and an MTA-peak but not an Mbd3 peak (Chd4+Mta-Mbd3 genes), or with an MTA-peak but not a Chd4 peak (MTA-only genes) were divided into quartiles based upon expression levels in wild type cells. Change in gene expression is plotted on the x-axis and calculated significance of the expression change on the y-axis. Those genes significantly misexpressed (|log2FC|>1; p≤0.05) are indicated in red, genes showing no significant change are plotted in black. The percentage of genes within each quartile which is significantly misexpressed is indicated at the top of each plot.


Phase contrast pictures of ES cells of indicated genotypes after 5 days in neuroectoderm differentiation conditions ( N2B27 ) . White arrowheads in the Mbd3 ∆ panel indicate pockets of ES - like cells . Scale bars represent 100 µm .

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Phase contrast pictures of ES cells of indicated genotypes after 5 days in neuroectoderm differentiation conditions (N2B27). White arrowheads in the Mbd3∆ panel indicate pockets of ES-like cells. Scale bars represent 100 µm.


Expression of indicated genes in the neuroectoderm differentiation time course was measured by RT - qPCR in Control ( green ) Mta123 ∆ ( magenta ) or Mbd3 ∆ ( blue ) ES cells . N ≥ 3 biological replicates , error bars indicate SEM .

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Expression of indicated genes in the neuroectoderm differentiation time course was measured by RT-qPCR in Control (green) Mta123∆ (magenta) or Mbd3∆ (blue) ES cells. N ≥ 3 biological replicates, error bars indicate SEM.


Phase contrast images of embryoid body outgrowths of indicated genotypes after 12 days in differentiation conditions ( N2B27 + 10 % serum ) . Scale bars represent 100µm .

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Phase contrast images of embryoid body outgrowths of indicated genotypes after 12 days in differentiation conditions (N2B27 + 10% serum). Scale bars represent 100µm.


Expression of indicated genes in embryoid body cultures over time was measured by RT -

Expression of indicated genes in embryoid body cultures over time was measured by RT-qPCR


Comparison of expression data for wild type , Mbd3 ∆ or Mta123 ∆ ES cells in self - renewing ( 2i ) conditions or after 48 hours in differentiation conditions ( 48h N2B27 ) with mouse embryonic single cell RNA - seq data Larger shading encloses biological replicates , smaller circles represent individual cells .

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Comparison of expression data for wild type, Mbd3∆ or Mta123∆ ES cells in self-renewing (2i) conditions or after 48 hours in differentiation conditions (48h N2B27) with mouse embryonic single cell RNA-seq data Larger shading encloses biological replicates, smaller circles represent individual cells.


Composite images of representative chimaeric embryos made with control ( Mta1Flox / ∆ Mta2 + / + Mta3Flox / Flox ) or Mta123 ∆ ES cells . ES - derived cells express the Kusabira Orange fluorescent marker . Sox2 indicates epiblast cells and Cdx2 is expressed in trophectoderm cells . Arrows indicate examples of K - Orange expressing cells in the mutant embryos . Scale bars = 20µm .

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Composite images of representative chimaeric embryos made with control (Mta1Flox/∆Mta2+/+Mta3Flox/Flox) or Mta123∆ ES cells. ES-derived cells express the Kusabira Orange fluorescent marker. Sox2 indicates epiblast cells and Cdx2 is expressed in trophectoderm cells. Arrows indicate examples of K-Orange expressing cells in the mutant embryos. Scale bars = 20µm.


( Top ) Number of K - Orange expressing cells observed in chimaeric embryos obtained using control ES cells , Mta123 ∆ ES cells , or Mta123 ∆ ES cells in which Mta2 was reintroduced on a constitutively expressed transgene ( Mta123 ∆ + Mta2TG ) . P - values calculated using a two - tailed t - test . ( Middle ) Mean number of K - Orange cells per embryo separated by Sox2 expression . P - values calculated using a two - tailed t - test . ( Bottom ) Number of K - Orange and Caspase - 3 positive cells per embryo . P - values calculated using a one - tailed t - test : * P < 0 . 05 , * * * * P < 0 . 0001 , " ns " = not significant .

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(Top) Number of K-Orange expressing cells observed in chimaeric embryos obtained using control ES cells, Mta123∆ ES cells, or Mta123∆ ES cells in which Mta2 was reintroduced on a constitutively expressed transgene (Mta123∆+Mta2TG). P-values calculated using a two-tailed t-test. (Middle) Mean number of K-Orange cells per embryo separated by Sox2 expression. P-values calculated using a two-tailed t-test. (Bottom) Number of K-Orange and Caspase-3 positive cells per embryo. P-values calculated using a one-tailed t-test: *P < 0.05, ****P < 0.0001, "ns" = not significant.


RNAPII - RBP - RNA complex was immunoprecipitated by anti - RNAPII antibody or control antibody ( cont ) , and subjected to immunoblotting . The input lanes contained 0 . 4 % of lysates used in the immunoprecipitation experiments .

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RNAPII-RBP-RNA complex was immunoprecipitated by anti-RNAPII antibody or control antibody (cont), and subjected to immunoblotting. The input lanes contained 0.4% of lysates used in the immunoprecipitation experiments.


Generation of tRIP - seq libraries of RNAPII - RBP - RNA complexes . After UV crosslinking , protein - RNA complexes were immunoprecipitated with anti - RNAPII ( 1st IP ) . RNA of the isolated RNAPII - RBP - RNA complexes was partially digested with RNase on beads , and RBP - RNA complexes dissociated from the bead were further immunoprecipitated with anti - FUS or anti - U1C antibody bound to the other beads ( 2nd IP ) . The immunoprecipitated FUS - RNA or U1C - RNA complex was treated with TEX , and then subjected to tRIP - seq library generation . The bottom panels show PCR - amplification of tRIP - seq libraries generated from RNAPII - FUS - RNA complex ( left panel ) and RNAPII - U1C - RNA complex ( right panel ) .

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Generation of tRIP-seq libraries of RNAPII-RBP-RNA complexes. After UV crosslinking, protein-RNA complexes were immunoprecipitated with anti-RNAPII (1st IP). RNA of the isolated RNAPII-RBP-RNA complexes was partially digested with RNase on beads, and RBP-RNA complexes dissociated from the bead were further immunoprecipitated with anti-FUS or anti-U1C antibody bound to the other beads (2nd IP). The immunoprecipitated FUS-RNA or U1C-RNA complex was treated with TEX, and then subjected to tRIP-seq library generation. The bottom panels show PCR-amplification of tRIP-seq libraries generated from RNAPII-FUS-RNA complex (left panel) and RNAPII-U1C-RNA complex (right panel).


PolyA - seq was performed using N2A cells to examine the effect of downregulation of FUS and U1 snRNP on polyA site usage . Cells were treated with siRNA against Fus ( siFus ) and / or U1 antisense morpholino ( U1AS ) . Control cells ( Cont ) were treated with both control siRNA and control antisense morpholino . A . Correlation of changes in polyA site usage between U1 - inhibition and Fus - silencing . Fold change in the RPM of a polyA site between siFus and Cont ( siFus / Cont ) was plotted against that between U1AS and Cont ( U1AS / Cont ) . Pearson ' s correlation coefficient ( r ) and the slope of the regression line are shown . B . Correlation of changes in polyA site usage between U1 - inhibition alone and the combination of Fus - silencing and U1 - inhibition . Fold change in the RPM of a polyA site between siFus + U1AS and Cont ( siFus + U1AS / Cont ) was plotted against that between U1AS and Cont ( U1AS / Cont ) . Pearson ' s correlation coefficient ( r ) and the slope of the regression line are shown . A slope of 1 . 0 indicates that Fus - silencing has no additive effect on U1 - inhibition .

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

PolyA-seq was performed using N2A cells to examine the effect of downregulation of FUS and U1 snRNP on polyA site usage. Cells were treated with siRNA against Fus (siFus) and/or U1 antisense morpholino (U1AS). Control cells (Cont) were treated with both control siRNA and control antisense morpholino. A. Correlation of changes in polyA site usage between U1-inhibition and Fus-silencing. Fold change in the RPM of a polyA site between siFus and Cont (siFus/Cont) was plotted against that between U1AS and Cont (U1AS/Cont). Pearson's correlation coefficient (r) and the slope of the regression line are shown. B. Correlation of changes in polyA site usage between U1-inhibition alone and the combination of Fus-silencing and U1-inhibition. Fold change in the RPM of a polyA site between siFus+U1AS and Cont (siFus+U1AS/Cont) was plotted against that between U1AS and Cont (U1AS/Cont). Pearson's correlation coefficient (r) and the slope of the regression line are shown. A slope of 1.0 indicates that Fus-silencing has no additive effect on U1-inhibition.


Localization of FUS in R495X _ N2A cells ( R495X ) , and wild - type N2A cells ( WT ) . Cells were fractionated to separate nucleus and cytoplasm . Equal proportion of cell extracts from nuclear and cytoplasmic fractions were immunoblotted with indicated antibodies .

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Localization of FUS in R495X_N2A cells (R495X), and wild-type N2A cells (WT). Cells were fractionated to separate nucleus and cytoplasm. Equal proportion of cell extracts from nuclear and cytoplasmic fractions were immunoblotted with indicated antibodies.


Interactions between FUS and U1 snRNP in the R495X _ N2A cells ( R495X ) and wild - type N2A cells ( WT ) . Total cell lysates were immunoprecipitated with anti - U1 - 70K antibody or a control antibody ( cont ) , and immunoblotted with indicated antibodies .

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Interactions between FUS and U1 snRNP in the R495X_N2A cells (R495X) and wild-type N2A cells (WT). Total cell lysates were immunoprecipitated with anti-U1-70K antibody or a control antibody (cont), and immunoblotted with indicated antibodies.


Effects of the R495X mutation on four categories of polyA sites . Four categories of polyA sites are comprised of ( 1 ) FUS - repressed ( rep ) / U1 - repressed sites , ( 2 ) FUS - activated ( act ) / U1 - repressed sites , ( 3 ) FUS - repressed / U1 - activated sites , and ( 4 ) FUS - activated / U1 - activated sites , according to Fig 4A . Fold change of each polyA site usage by the R495X mutation ( R495X / WT ) is shown in violin plots . Bars indicate the median and 95 % confidential intervals for each category . * * * p < 0 . 001 , * * * * p < 0 . 0001 , based on the Steel - Dwass test for comparing all 6 pairs of categories .

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Effects of the R495X mutation on four categories of polyA sites. Four categories of polyA sites are comprised of (1) FUS-repressed (rep) /U1-repressed sites, (2) FUS-activated (act) /U1-repressed sites, (3) FUS-repressed/U1-activated sites, and (4) FUS-activated/U1-activated sites, according to Fig 4A. Fold change of each polyA site usage by the R495X mutation (R495X/WT) is shown in violin plots. Bars indicate the median and 95% confidential intervals for each category. *** p < 0.001, **** p < 0.0001, based on the Steel-Dwass test for comparing all 6 pairs of categories.


A . qPCR analysis of Ahr expression in B cell subsets purified from C57Bl / 6 mice . Ahr expression was normalized to Hprt1 . n = 3 - 5 independent experiments ; mean ± SD .

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A. qPCR analysis of Ahr expression in B cell subsets purified from C57Bl/6 mice. Ahr expression was normalized to Hprt1. n = 3-5 independent experiments; mean ± SD.


B . qPCR analysis of Ahr expression in splenic CD19 + cells isolated from C57Bl / 6 mice and cultured for 4h as indicated . Ahr expression was normalized to Hprt1 . Ahr expression among groups was normalized to Medium . n = 4 independent experiments ; mean ± SEM ; one - way ANOVA , Dunnett ' s multiple comparison test .

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B. qPCR analysis of Ahr expression in splenic CD19+ cells isolated from C57Bl/6 mice and cultured for 4h as indicated. Ahr expression was normalized to Hprt1. Ahr expression among groups was normalized to Medium. n = 4 independent experiments; mean ± SEM; one-way ANOVA, Dunnett's multiple comparison test.


C . qPCR analysis of Ahr expression in splenic CD19 + cells isolated from C57Bl / 6 mice and cultured for 4h with 20 ng / ml IL - 4 and / or 10 μg / ml α - IgM . Ahr expression was normalized to Hprt1 . Ahr expression among groups was normalized to Medium . n = 3 independent experiments ; mean ± SEM ; one - way ANOVA , Tukey ' s multiple comparison test .

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C. qPCR analysis of Ahr expression in splenic CD19+ cells isolated from C57Bl/6 mice and cultured for 4h with 20 ng/ml IL-4 and/or 10 μg/ml α-IgM. Ahr expression was normalized to Hprt1. Ahr expression among groups was normalized to Medium. n = 3 independent experiments; mean ± SEM; one-way ANOVA, Tukey's multiple comparison test.


D . Western blot of protein extracts from splenic CD19 + cells isolated from C57Bl / 6 mice and cultured for 4h with 20 ng / ml IL - 4 and / or 10 μg / ml α - IgM . Values above the blots indicate AhR protein quantification obtained by densitometry , normalized to β - actin and compared to Medium sample . Sample A and B indicate 2 independent replicates .

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D. Western blot of protein extracts from splenic CD19+ cells isolated from C57Bl/6 mice and cultured for 4h with 20 ng/ml IL-4 and/or 10 μg/ml α-IgM. Values above the blots indicate AhR protein quantification obtained by densitometry, normalized to β-actin and compared to Medium sample. Sample A and B indicate 2 independent replicates.


E . Quantification by densitometric analysis of the results shown in C . n = 2 independent experiments ; mean ± range .

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E. Quantification by densitometric analysis of the results shown in C. n = 2 independent experiments; mean ± range.


F . qPCR analysis of Ahr expression in purified splenic B cell subsets isolated from C57Bl / 6 mice and cultured as indicated for 4h . Ahr expression was normalized to Hprt1 . n = 2 independent experiments ; mean ± range .

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F. qPCR analysis of Ahr expression in purified splenic B cell subsets isolated from C57Bl/6 mice and cultured as indicated for 4h. Ahr expression was normalized to Hprt1. n = 2 independent experiments; mean ± range.


G . qPCR analysis of Ahr expression in splenic CD19 + cells isolated from C57Bl / 6 mice and cultured for the indicated time points with 20 ng / ml IL - 4 and / or 10 μg / ml α - IgM . Ahr expression was normalized to Hprt1 . Ahr expression among groups was normalized to Medium . n = 5 independent experiments ; mean ± SEM ; one - way ANOVA , Dunnett ' s multiple comparison test .

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G. qPCR analysis of Ahr expression in splenic CD19+ cells isolated from C57Bl/6 mice and cultured for the indicated time points with 20 ng/ml IL-4 and/or 10 μg/ml α-IgM. Ahr expression was normalized to Hprt1. Ahr expression among groups was normalized to Medium. n = 5 independent experiments; mean ± SEM; one-way ANOVA, Dunnett's multiple comparison test.


A . Western blot of nuclear and cytoplasmic protein extracts from splenic CD19 + cells isolated from C57Bl / 6 mice and cultured with anti - IgM ( α - IgM ) for 4h , followed by the indicated treatment for 2h . Values above the blots indicate AhR protein quantification obtained by densitometry , normalized to Sam68 or Tubulin and compared to the DMSO - treated sample . Representative data of n = 3 independent experiments .

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A. Western blot of nuclear and cytoplasmic protein extracts from splenic CD19+ cells isolated from C57Bl/6 mice and cultured with anti-IgM (α-IgM) for 4h, followed by the indicated treatment for 2h. Values above the blots indicate AhR protein quantification obtained by densitometry, normalized to Sam68 or Tubulin and compared to the DMSO-treated sample. Representative data of n = 3 independent experiments.


B . qPCR analysis of Cyp1a1 expression in splenic CD19 + cells isolated from C57Bl / 6 mice and cultured for 24h as indicated . Cyp1a1 expression was normalized to Hprt1 . Cyp1a1 expression among groups was normalized to Medium DMSO . n = 2 independent experiments ; mean ± range .

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B. qPCR analysis of Cyp1a1 expression in splenic CD19+ cells isolated from C57Bl/6 mice and cultured for 24h as indicated. Cyp1a1 expression was normalized to Hprt1. Cyp1a1 expression among groups was normalized to Medium DMSO. n = 2 independent experiments; mean ± range.


C . Flow cytometry analysis of Cyp1a1 expression , reported by eYFP , in splenic CD19 + cells isolated from Cyp1a1Cre R26R eYFP mice and cultured for 72h as indicated . Representative data of n = 3 independent experiments .

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C. Flow cytometry analysis of Cyp1a1 expression, reported by eYFP, in splenic CD19+ cells isolated from Cyp1a1Cre R26R eYFP mice and cultured for 72h as indicated. Representative data of n = 3 independent experiments.


D . qPCR analysis of Cyp1a1 expression in purified splenic B cell subsets isolated from C57Bl / 6 mice and cultured as indicated for 24h . Cyp1a1 expression was normalized to Hprt1 . n = 2 independent experiments ; mean ± range .

D. qPCR analysis of Cyp1a1 expression in purified splenic B cell subsets isolated from C57Bl/6 mice and cultured as indicated for 24h. Cyp1a1 expression was normalized to Hprt1. n = 2 independent experiments; mean ± range.


B . Representative flow cytometry analysis of Cyp1a1 expression , reported by eYFP , in CD45 . 1 + cells harvested from mice challenged as in A . Gating is indicated above the plots . Representative data of n = 3 independent experiments . GC cells : CD95 + GL - 7 + ; non - GC cells : CD95 - GL - 7 - . C , D . Summary of percentage of GC ( C ) and non - GC eYFP + ( D ) cells from flow cytometry analysis . n = 3 independent experiments ; lines indicate mean ; one - way ANOVA , Tukey ' s multiple comparison test .

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B. Representative flow cytometry analysis of Cyp1a1 expression, reported by eYFP, in CD45.1+ cells harvested from mice challenged as in A. Gating is indicated above the plots. Representative data of n = 3 independent experiments. GC cells: CD95+ GL-7+; non-GC cells: CD95- GL-7-.C, D. Summary of percentage of GC (C) and non-GC eYFP+ (D) cells from flow cytometry analysis. n = 3 independent experiments; lines indicate mean; one-way ANOVA, Tukey's multiple comparison test.


A . Flow cytometry analysis of CTV dilution in splenic CD19 + cells sorted from non - immune Ahrfl / + mb1Cre + ( Ahr + / - B cells , solid grey ) and Ahrfl / - mb1Cre + ( Ahr - / - B cells , black ) mice stimulated for 72h as indicated . Treatment of Ahr + / - CD19 + cells with CH223191 is indicated in red . Representative data of n = 4 - 5 independent experiments . B - D . Flow cytometry analysis of expansion index ( B ) , % of divided cells ( C ) and replication index ( D ) of splenic CD19 + cells sorted from non - immune Ahrfl / + mb1Cre + ( black ) and Ahrfl / - mb1Cre + ( white ) mice stimulated for 72h as indicated . Treatment of Ahr + / - CD19 + cells with CH223191 is indicated in red . n = 4 - 5 independent experiments ; mean ± SEM ; 2 - way ANOVA , Sidak ' s multiple comparison test .

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A. Flow cytometry analysis of CTV dilution in splenic CD19+ cells sorted from non-immune Ahrfl/+mb1Cre+ (Ahr+/- B cells, solid grey) and Ahrfl/-mb1Cre+ (Ahr-/- B cells, black) mice stimulated for 72h as indicated. Treatment of Ahr+/- CD19+ cells with CH223191 is indicated in red. Representative data of n = 4-5 independent experiments.B-D. Flow cytometry analysis of expansion index (B), % of divided cells (C) and replication index (D) of splenic CD19+ cells sorted from non-immune Ahrfl/+mb1Cre+ (black) and Ahrfl/-mb1Cre+ (white) mice stimulated for 72h as indicated. Treatment of Ahr+/- CD19+ cells with CH223191 is indicated in red. n = 4-5 independent experiments; mean ± SEM; 2-way ANOVA, Sidak's multiple comparison test.


E . Flow cytometry analysis of cell cycle distribution of splenic CD19 + cells purified from non - immune Ahrfl / + mb1Cre + ( black ) and Ahrfl / - mb1Cre + ( white ) mice stimulated for 48h with 5 µg / ml α - IgM . Data representative of n = 2 independent experiments ; lines indicate mean ; 2 - way ANOVA , Sidak ' s multiple comparison test .

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E. Flow cytometry analysis of cell cycle distribution of splenic CD19+ cells purified from non-immune Ahrfl/+mb1Cre+ (black) and Ahrfl/-mb1Cre+ (white) mice stimulated for 48h with 5 µg/ml α-IgM. Data representative of n = 2 independent experiments; lines indicate mean; 2-way ANOVA, Sidak's multiple comparison test.


A , B . Flow cytometry analysis of distribution of CD45 . 1 + and CD45 . 2 + cells in indicated cell subsets purified from bone marrow ( A ) and lymph node ( B ) of sub lethally - irradiated Rag1 - / - mice 8 weeks after reconstitution with equal numbers of bone marrow cells from Ahr + / + ( CD45 . 1 + ) and Ahr - / - ( CD45 . 2 + ) mice . Dashed lines indicate 50 % level . Representative data of n = 2 independent experiments ; two - tailed paired t test .

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A, B. Flow cytometry analysis of distribution of CD45.1+ and CD45.2+ cells in indicated cell subsets purified from bone marrow (A) and lymph node (B) of sub lethally-irradiated Rag1-/- mice 8 weeks after reconstitution with equal numbers of bone marrow cells from Ahr+/+ (CD45.1+) and Ahr-/- (CD45.2+) mice. Dashed lines indicate 50% level. Representative data of n = 2 independent experiments; two-tailed paired t test.


D . Flow cytometry analysis of distribution of CD45 . 1 + CD45 . 2 + and CD45 . 2 + cells harvested from host mice challenged as indicated above the dot plots . Representative data of n = 3 independent experiments .

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D. Flow cytometry analysis of distribution of CD45.1+CD45.2+ and CD45.2+ cells harvested from host mice challenged as indicated above the dot plots. Representative data of n = 3 independent experiments.


E . Flow cytometry analysis of distribution of Ahr + / + CD45 . 1 + CD45 . 2 + ( black ) and Ahr - / - CD45 . 2 + ( white ) cells harvested from host mice . Indicated distribution is quantified relative to transferred cells . X symbols indicate SRBC - mock - treated control . Dashed line indicates 50 % threshold . Representative data of n = 3 independent experiments ; two - tailed paired t test .

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E. Flow cytometry analysis of distribution of Ahr+/+ CD45.1+CD45.2+ (black) and Ahr-/- CD45.2+ (white) cells harvested from host mice. Indicated distribution is quantified relative to transferred cells. X symbols indicate SRBC-mock-treated control. Dashed line indicates 50% threshold. Representative data of n = 3 independent experiments; two-tailed paired t test.


F , G . Flow cytometry analysis of distribution ( F ) and cell numbers ( G ) of IgG1 + and IgG1 - HEL3x - binding Ahr + / + CD45 . 1 + CD45 . 2 + ( black ) and Ahr - / - CD45 . 2 + ( white ) cells harvested from host mice . Representative data of n = 2 independent experiments ; two - tailed paired t test .

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F, G. Flow cytometry analysis of distribution (F) and cell numbers (G) of IgG1+ and IgG1- HEL3x-binding Ahr+/+ CD45.1+CD45.2+ (black) and Ahr-/- CD45.2+ (white) cells harvested from host mice. Representative data of n = 2 independent experiments; two-tailed paired t test.


A , B . Tables showing top 20 down - regulated ( A ) genes in Ahr - / - B cells as compared to Ahr + / + cells , after analysis by RNA sequencing . B cells were activated with 10 µg / ml α - IgM + 20 ng / ml IL - 4 for 8h ; 250 nM FICZ was added to the culture during the last 4h . The average read counts are directly proportional to the extent of expression of a given gene in Ahr + / + or Ahr - / - cells . Data from n = 3 mice per group . Average read counts for the housekeeping gene Hprt1 in both Ahr + / + and Ahr - / - B cells are indicated below the table in figure 6A .

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A, B. Tables showing top 20 down-regulated (A) genes in Ahr-/- B cells as compared to Ahr+/+ cells, after analysis by RNA sequencing. B cells were activated with 10 µg/ml α-IgM + 20 ng/ml IL-4 for 8h; 250 nM FICZ was added to the culture during the last 4h. The average read counts are directly proportional to the extent of expression of a given gene in Ahr+/+ or Ahr-/- cells. Data from n = 3 mice per group. Average read counts for the housekeeping gene Hprt1 in both Ahr+/+ and Ahr-/- B cells are indicated below the table in figure 6A.


A , B . Tables showing top up - regulated ( B ) genes in Ahr - / - B cells as compared to Ahr + / + cells , after analysis by RNA sequencing . B cells were activated with 10 µg / ml α - IgM + 20 ng / ml IL - 4 for 8h ; 250 nM FICZ was added to the culture during the last 4h . The average read counts are directly proportional to the extent of expression of a given gene in Ahr + / + or Ahr - / - cells . Data from n = 3 mice per group . Average read counts for the housekeeping gene Hprt1 in both Ahr + / + and Ahr - / - B cells are indicated below the table in figure 6A .

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A, B. Tables showing top up-regulated (B) genes in Ahr-/- B cells as compared to Ahr+/+ cells, after analysis by RNA sequencing. B cells were activated with 10 µg/ml α-IgM + 20 ng/ml IL-4 for 8h; 250 nM FICZ was added to the culture during the last 4h. The average read counts are directly proportional to the extent of expression of a given gene in Ahr+/+ or Ahr-/- cells. Data from n = 3 mice per group. Average read counts for the housekeeping gene Hprt1 in both Ahr+/+ and Ahr-/- B cells are indicated below the table in figure 6A.


C . qPCR analysis of Ccno expression in splenic CD19 + cells isolated from non - immune Ahrfl / + mb1Cre + ( Ahr + / - B cells , black ) and Ahrfl / - mb1Cre + ( Ahr - / - B cells , white ) mice , stimulated with 10 µg / ml anti - IgM for the indicated time points . Ccno expression was normalized to Hprt1 . n = 3 independent experiments ; mean ± SEM ; two - way ANOVA , Sidak ' s multiple comparison test .

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C. qPCR analysis of Ccno expression in splenic CD19+ cells isolated from non-immune Ahrfl/+mb1Cre+ (Ahr+/- B cells, black) and Ahrfl/-mb1Cre+ (Ahr-/- B cells, white) mice, stimulated with 10 µg/ml anti-IgM for the indicated time points. Ccno expression was normalized to Hprt1. n = 3 independent experiments; mean ± SEM; two-way ANOVA, Sidak's multiple comparison test.


D . qPCR analysis of Ccno expression in splenic CD19 + cells isolated from C57Bl / 6 mice and cultured for 8h with 20 ng / ml IL - 4 and / or 10 μg / ml α - IgM . Ccno expression was normalized to Hprt1 . n = 3 independent experiments ; mean ± SEM ; one - way ANOVA , Tukey ' s multiple comparison test .

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D. qPCR analysis of Ccno expression in splenic CD19+ cells isolated from C57Bl/6 mice and cultured for 8h with 20 ng/ml IL-4 and/or 10 μg/ml α-IgM. Ccno expression was normalized to Hprt1. n = 3 independent experiments; mean ± SEM; one-way ANOVA, Tukey's multiple comparison test.


E . qPCR analysis of Ccno expression in splenic CD19 + cells isolated from C57Bl / 6 mice and cultured for the indicated time points with 20 ng / ml IL - 4 and / or 10 μg / ml α - IgM in presence or absence of 250 nM FICZ or 1 μM 3 - MC . Ccno expression was normalized to Hprt1 . n = 3 independent experiments ; mean ± SEM ; one - way ANOVA , Tukey ' s multiple comparison test .

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E. qPCR analysis of Ccno expression in splenic CD19+ cells isolated from C57Bl/6 mice and cultured for the indicated time points with 20 ng/ml IL-4 and/or 10 μg/ml α-IgM in presence or absence of 250 nM FICZ or 1 μM 3-MC. Ccno expression was normalized to Hprt1. n = 3 independent experiments; mean ± SEM; one-way ANOVA, Tukey's multiple comparison test.


F . Chromatin Immunoprecipitation ( ChIP ) analysis of AhR interaction with the Ccno promoter and an irrelevant region ( - 3 . 3 Kb from Ccno transcription starting site ) in Ahr + / + ( black ) and Ahr - / - ( white ) B cells 5h after activation with 10 µg / ml α - IgM + 20 ng / ml IL - 4 . 250 nM FICZ was added in the last hour of culture . Representative data of n = 2 independent experiments ; mean ± SEM ; two - way ANOVA , Sidak ' s multiple comparison test .

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F. Chromatin Immunoprecipitation (ChIP) analysis of AhR interaction with the Ccno promoter and an irrelevant region (-3.3 Kb from Ccno transcription starting site) in Ahr+/+ (black) and Ahr-/- (white) B cells 5h after activation with 10 µg/ml α-IgM + 20 ng/ml IL-4. 250 nM FICZ was added in the last hour of culture. Representative data of n = 2 independent experiments; mean ± SEM; two-way ANOVA, Sidak's multiple comparison test.


A Tissue distribution of RIPK2 determined by anti - FLAG immunoprecipitation . Organ homogenates from WT ( wild - type ) and KI ( homozygous FLAG - RIPK2 ) mice were subjected to anti - FLAG immunoprecipitation and immunoblotting .

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A Tissue distribution of RIPK2 determined by anti-FLAG immunoprecipitation. Organ homogenates from WT (wild-type) and KI (homozygous FLAG-RIPK2) mice were subjected to anti-FLAG immunoprecipitation and immunoblotting.


B Inflammatory signaling in wild - type ( WT / WT ) , RIPK2 CRISPR KO ( KO / KO ) and FLAG - RIPK2 heterozygous ( WT / KI ) and homozygous ( KI / KI ) BMDMs . BMDMs were primed with IFNy , stimulated with MDP for indicated times and analysed by immunoblotting .

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B Inflammatory signaling in wild-type (WT/WT), RIPK2 CRISPR KO (KO/KO) and FLAG-RIPK2 heterozygous (WT/KI) and homozygous (KI/KI) BMDMs. BMDMs were primed with IFNy, stimulated with MDP for indicated times and analysed by immunoblotting.


C Cytokine production of RIPK2 CRISPR KO ( KO ) , wild - type ( WT ) and FLAG - RIPK2 heterozygous ( WT / KI ) and homozygous ( KI / KI ) BMDMs in response to MDP . BMDMs were left untreated or treated with IFNy or IFNy and MDP over - night and cytokines were measured by ELISA . N = 5 - 8 mice . Shown is average ± SEM . ns = P > 0 . 05 ; * * = P ≤ 0 . 01 ; * * * * = P ≤ 0 . 0001 ; two - way ANOVA .

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C Cytokine production of RIPK2 CRISPR KO (KO), wild-type (WT) and FLAG-RIPK2 heterozygous (WT/KI) and homozygous (KI/KI) BMDMs in response to MDP. BMDMs were left untreated or treated with IFNy or IFNy and MDP over-night and cytokines were measured by ELISA. N = 5-8 mice. Shown is average ± SEM. ns = P > 0.05; ** = P ≤ 0.01; **** = P ≤ 0.0001; two-way ANOVA.


D Serum cytokines in RIPK2 CRISPR KO ( KO ) , wild - type ( WT ) and FLAG - RIPK2 heterozygous ( WT / KI ) or homozygous ( KI / KI ) mice after i . p . MDP administration . Mice were injected i . p . with PBS or MDP , sacrificed after 4h and serum cytokines were measured by ELISA . N = 3 - 6 mice . Shown is average ± SEM . ns = P > 0 . 05 ; * * = P ≤ 0 . 01 ; two - way ANOVA .

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D Serum cytokines in RIPK2 CRISPR KO (KO), wild-type (WT) and FLAG-RIPK2 heterozygous (WT/KI) or homozygous (KI/KI) mice after i.p. MDP administration. Mice were injected i.p. with PBS or MDP, sacrificed after 4h and serum cytokines were measured by ELISA. N = 3-6 mice. Shown is average ± SEM. ns = P > 0.05; ** = P ≤ 0.01; two-way ANOVA.


A Two step enrichment to isolate ubiquitinated RIPK2 from BMDMs . Protein lysates from FLAG - RIPK2 BMDMs ( A ) were sequentially subjected to ubiquitin enrichment ( UBA , B ) and FLAG pulldown ( C ) prior to protein elution and subsequent mass spectrometry analysis .

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A Two step enrichment to isolate ubiquitinated RIPK2 from BMDMs. Protein lysates from FLAG-RIPK2 BMDMs (A) were sequentially subjected to ubiquitin enrichment (UBA, B) and FLAG pulldown (C) prior to protein elution and subsequent mass spectrometry analysis.


A Activation of the NF - κB pathway by RIPK2 Lysine - and phosphosite mutants . RIPK2 - deficient THP - 1 cells reconstituted with wild - type RIPK2 or RIPK2 mutants were stimulated with L18 - MDP , harvested at indicated time points and activation of the NF - κB pathway was analyzed by immunoblotting .

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

A Activation of the NF-κB pathway by RIPK2 Lysine- and phosphosite mutants. RIPK2-deficient THP-1 cells reconstituted with wild-type RIPK2 or RIPK2 mutants were stimulated with L18-MDP, harvested at indicated time points and activation of the NF-κB pathway was analyzed by immunoblotting.


B IL - 8 production of wild - type THP - 1 and RIPK2 - deficient THP - 1 cells reconstituted with wild - type RIPK2 or RIPK2 mutants and stimulated with L18 - MDP was assessed by ELISA . N = 4 - 8 experiments . Shown is average ± SEM . * = P ≤ 0 . 05 ; two - way ANOVA .

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

B IL-8 production of wild-type THP-1 and RIPK2-deficient THP-1 cells reconstituted with wild-type RIPK2 or RIPK2 mutants and stimulated with L18-MDP was assessed by ELISA. N = 4-8 experiments. Shown is average ± SEM. * = P ≤ 0.05; two-way ANOVA.


C ) RIPK2 ubiquitination determined by UBA pulldown in RIPK2 - deficient cells reconstituted with wild - type or mutant RIPK2 after stimulation with L18 - MDP .

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

C) RIPK2 ubiquitination determined by UBA pulldown in RIPK2-deficient cells reconstituted with wild-type or mutant RIPK2 after stimulation with L18-MDP.


D ) Detection of K63 - and M1 - linked ubiquitin chains by UBA pulldown or pulldown with ubiquitin chain type - specific antibodies in RIPK2 - deficient cells reconstituted with RIPK2 K209R after stimulation with L18 - MDP .

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

D) Detection of K63- and M1-linked ubiquitin chains by UBA pulldown or pulldown with ubiquitin chain type-specific antibodies in RIPK2-deficient cells reconstituted with RIPK2 K209R after stimulation with L18-MDP.


C Activation of the NF - κB pathway by RIPK2 pocket mutants . RIPK2 - deficient THP - 1 cells were reconstituted with wild - type RIPK2 or RIPK2 mutants , stimulated with L18 - MDP , harvested at indicated time points and analyzed by immunoblotting .

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

C Activation of the NF-κB pathway by RIPK2 pocket mutants. RIPK2-deficient THP-1 cells were reconstituted with wild-type RIPK2 or RIPK2 mutants, stimulated with L18-MDP, harvested at indicated time points and analyzed by immunoblotting.


D IL - 8 production of RIPK2 - deficient THP - 1cells reconstituted with wild - type RIPK2 or RIPK2 mutants and stimulated with L18 - MDP was assessed by ELISA . N = 3 - 8 experiments . Shown is average ± SEM . ns = P > 0 . 05 ; * = P ≤ 0 . 05 ; * * * = P ≤ 0 . 001 ; * * * * = P ≤ 0 . 0001 ; two - way ANOVA .

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

D IL-8 production of RIPK2-deficient THP-1cells reconstituted with wild-type RIPK2 or RIPK2 mutants and stimulated with L18-MDP was assessed by ELISA. N = 3-8 experiments. Shown is average ± SEM. ns = P > 0.05; * = P ≤ 0.05; *** = P ≤ 0.001; **** = P ≤ 0.0001; two-way ANOVA.


E Ubiquitination of RIPK2 pocket mutants . RIPK2 - deficient THP - 1 cells were reconstituted with wild - type or mutant RIPK2 , left unstimulated or stimulated with L18 - MDP and subjected to UBA pulldown and immunoblotting .

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

E Ubiquitination of RIPK2 pocket mutants. RIPK2-deficient THP-1 cells were reconstituted with wild-type or mutant RIPK2, left unstimulated or stimulated with L18-MDP and subjected to UBA pulldown and immunoblotting.


A : Thermal stability assay using selected RIPK2 mutants . THP - 1 cells expressing RIPK2 were subjected to heat treatment and the non - denatured fraction was analysed by immunoblotting .

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

A: Thermal stability assay using selected RIPK2 mutants. THP-1 cells expressing RIPK2 were subjected to heat treatment and the non-denatured fraction was analysed by immunoblotting.


B : Binding of RIPK2 to XIAP - BIR2 . Lysates from RIPK2 - deficient THP - 1 cells reconstituted with WT or mutant RIPK2 were subjected to pulldown experiments with recombinantly expressed XIAP - BIR2 and analysed by immunoblotting .

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

B: Binding of RIPK2 to XIAP-BIR2. Lysates from RIPK2-deficient THP-1 cells reconstituted with WT or mutant RIPK2 were subjected to pulldown experiments with recombinantly expressed XIAP-BIR2 and analysed by immunoblotting.


( A ) Illustration of the crossing and ageing scheme used to obtain total RNA extracts from fly heads for the transcriptional profiling and all successive qPCR assays . Expression of different Atrophin forms with the GMR driver was induced , owing to a temperature ‐ sensitive mutant Gal80 repressor . F1 flies were allowed to develop at 18 ° C ; at this temperature the Gal80 repressor keeps transgenes silent . Newly eclosed flies ( 0 - 48 h ) were collected and killed immediately ( 0d ) or aged for 2 or 14 days at 29 ° C . This inactivates Gal80 and transgenes are switched on by GMR ‐ Gal4 . This protocol allows comparing siblings that differ exclusively in their age and transgenes expression . Control flies crossed to GMR ‐ Gal4 ; UbiGal80ts in all experiments are from the w1118 stock in which all UAS transgenes have been generated .

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

(A) Illustration of the crossing and ageing scheme used to obtain total RNA extracts from fly heads for the transcriptional profiling and all successive qPCR assays. Expression of different Atrophin forms with the GMR driver was induced, owing to a temperature‐sensitive mutant Gal80 repressor. F1 flies were allowed to develop at 18°C; at this temperature the Gal80 repressor keeps transgenes silent. Newly eclosed flies (0-48 h) were collected and killed immediately (0d) or aged for 2 or 14 days at 29°C. This inactivates Gal80 and transgenes are switched on by GMR‐Gal4. This protocol allows comparing siblings that differ exclusively in their age and transgenes expression. Control flies crossed to GMR‐Gal4; UbiGal80ts in all experiments are from the w1118 stock in which all UAS transgenes have been generated.


( B ) Tangential eye sections of flies representative of all the different populations used in the microarray analysis at all different time points . Weak degeneration is only visible after 14 days with polyQ Atro ; in particular with Atro75QN there is an initial loss of photoreceptors ( PR , arrow ) , 30 . 7 % of the ommatidia has lost at least 1 PR , that is only 5 . 1 % of all neuronal PR have been lost at this stage ( N = 333 ) .

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(B) Tangential eye sections of flies representative of all the different populations used in the microarray analysis at all different time points. Weak degeneration is only visible after 14 days with polyQ Atro; in particular with Atro75QN there is an initial loss of photoreceptors (PR, arrow), 30.7% of the ommatidia has lost at least 1 PR, that is only 5.1% of all neuronal PR have been lost at this stage (N=333).


( A ) Heat maps for three subsets of the pattern clustering analysis generated using the Euclidean Distance metric . Upregulated genes are in red and downregulated genes are in green . The downregulating transcriptional activity increases with time . The main branch in clustering of the different Atro mutants is along time points . See also Supplementary Figure 4 .

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(A) Heat maps for three subsets of the pattern clustering analysis generated using the Euclidean Distance metric. Upregulated genes are in red and downregulated genes are in green. The downregulating transcriptional activity increases with time. The main branch in clustering of the different Atro mutants is along time points. See also Supplementary Figure 4.


( A ) qPCR analysis of the fold changes ± s . d . of ft transcription . Downregulation starts at 2 days and progresses at 14 days .

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(A) qPCR analysis of the fold changes±s.d. of ft transcription. Downregulation starts at 2 days and progresses at 14 days.


( B ) qPCR analysis of the enrichment ± s . d . of different regions of the ft regulatory elements in ChIP for Atro from BG3neuronal cell extracts . Enrichment is calculated as the percentage of DNAimmunoprecipitated from extracts of cells in which Atro expression has been induced , with respect to the amount immunoprecipitated from uninduced cell extracts according to the ( Atro - No Ab ) induced / ( Atro − No Ab ) uninduced formula . The DNA region from IV chromosome has been previously shown not to be immunoprecipitated in ChIP for Atro ( Haecker et al , 2007 ) and has been used as a negative control .

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

(B) qPCR analysis of the enrichment±s.d. of different regions of the ft regulatory elements in ChIP for Atro from BG3neuronal cell extracts. Enrichment is calculated as the percentage of DNAimmunoprecipitated from extracts of cells in which Atro expression has been induced, with respect to the amount immunoprecipitated from uninduced cell extracts according to the (Atro-No Ab)induced/(Atro−No Ab)uninduced formula. The DNA region from IV chromosome has been previously shown not to be immunoprecipitated in ChIP for Atro ( Haecker et al, 2007) and has been used as a negative control.


( C ) Histograms showing the number of ommatidia with a full complement ( 7 ) of PR in flies expressing Atro75QN with the Rhodopsin1 driver in either a control ( w1118 ) or different mutant backgrounds and aged at 29 ° C for 28 days . Heterozygosis for two independent ft alleles and a sav allele significantly enhances the loss of PR . Mild overexpression of Wts via a GMR - wts transgene , which does not display any strong phenotype per se , significantly suppresses the loss of PR . No interaction was detected in this assay with wtsx1 and ykiB5 alleles in heterozygosity . N = 430 - 963 from at least four eyes .

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

(C) Histograms showing the number of ommatidia with a full complement (7) of PR in flies expressing Atro75QN with the Rhodopsin1 driver in either a control (w1118) or different mutant backgrounds and aged at 29°C for 28 days. Heterozygosis for two independent ft alleles and a sav allele significantly enhances the loss of PR. Mild overexpression of Wts via a GMR-wts transgene, which does not display any strong phenotype per se, significantly suppresses the loss of PR. No interaction was detected in this assay with wtsx1 and ykiB5 alleles in heterozygosity. N=430-963 from at least four eyes.


( D ) Tangential eye sections and histograms showing the degeneration of GMR > Atro75QN , Ubi - Gal80ts flies in combination with different UAS transgenes and aged 14 days as in Figure 1A . UAS - EGFP is used as a negative control . Either Ft or Wts overexpression strongly suppresses loss of PR caused by Atro75QN . Lower panels are provided as examples of quantification . A more modest but still significant rescue is observed by overexpressing a Yki RNAi contruct or Hpo ; however , in this case it is to be considered that the overexpression of Hpoper se brings about the loss of at least one PR in ∼ 5 % of ommatidia ( data not shown ) . No effect is detected with overexpression of Sav . Two ‐ tailed t ‐ test : * P0 . 05 ; * * P0 . 01 . The wtsEPG4808 is a previously uncharacterised EP element insertion at the 5 ′ of the wts gene . Rare homozygous escapers display larger eyes , whereas in combination with GMR - GMR this line gives rise to smaller rough eyes , and , finally , GMR analysis of GMR > wtsEPG4808 indicates a 10 ‐ fold increase in the head content of wts mRNA ( data not shown ) .

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

(D) Tangential eye sections and histograms showing the degeneration of GMR>Atro75QN, Ubi-Gal80ts flies in combination with different UAS transgenes and aged 14 days as in Figure 1A. UAS-EGFP is used as a negative control. Either Ft or Wts overexpression strongly suppresses loss of PR caused by Atro75QN. Lower panels are provided as examples of quantification. A more modest but still significant rescue is observed by overexpressing a Yki RNAi contruct or Hpo; however, in this case it is to be considered that the overexpression of Hpoper se brings about the loss of at least one PR in ∼5% of ommatidia (data not shown). No effect is detected with overexpression of Sav. Two‐tailed t‐test: *P0.05; **P0.01. The wtsEPG4808 is a previously uncharacterised EP element insertion at the 5′ of the wts gene. Rare homozygous escapers display larger eyes, whereas in combination with GMR-GMR this line gives rise to smaller rough eyes, and, finally, GMR analysis of GMR>wtsEPG4808 indicates a 10‐fold increase in the head content of wts mRNA (data not shown).


( A ) Tangential eye sections through ftfd clones aged 1 , 7 and 14 days , of ftfd , dDGC13 double mutant clones and of ftfd clones in a ykiB5 heterozygous background aged 14 days . Clones are marked by the absence of yellow pigment . Arrows point at degenerating photoreceptors , arrowheads point at intact wt photoreceptors in mosaic ommatidia . The circled ommatidium is genetically wt but non ‐ autonomously flipped in its polarity and has not degenerated . Lower panels are masks that show clonal borders and quantification of PR number per ommatidia . After 14 days , almost all ftfd cells have degenerated , whereas almost all ftfd , dDGC13 and some ftfd , ykiB5 / + neuronalphotoreceptors have not .

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

(A) Tangential eye sections through ftfd clones aged 1, 7 and 14 days, of ftfd,dDGC13 double mutant clones and of ftfd clones in a ykiB5 heterozygous background aged 14 days. Clones are marked by the absence of yellow pigment. Arrows point at degenerating photoreceptors, arrowheads point at intact wt photoreceptors in mosaic ommatidia. The circled ommatidium is genetically wt but non‐autonomously flipped in its polarity and has not degenerated. Lower panels are masks that show clonal borders and quantification of PR number per ommatidia. After 14 days, almost all ftfd cells have degenerated, whereas almost all ftfd,dDGC13 and some ftfd,ykiB5/+ neuronalphotoreceptors have not.


( B ) Histograms showing the quantification of degeneration in ftfd , ftfddDGC13 and ftfdykiB5 / + clones . Loss of ft leads to statistically significant loss of neurons and this is suppressed at 14 days by ykiB5and much more dramatically by dDGC13 . * * * P0 . 001 , * * P0 . 01 ; * P0 . 05 in two ‐ tailed t ‐ test .

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

(B) Histograms showing the quantification of degeneration in ftfd, ftfddDGC13 and ftfdykiB5/+ clones. Loss of ft leads to statistically significant loss of neurons and this is suppressed at 14 days by ykiB5and much more dramatically by dDGC13. ***P0.001, **P0.01; *P0.05 in two‐tailed t‐test.


( C ) Degeneration in mosaic ommatidia in ftfd clones . At this stage of degeneration approximately half of the non ‐ pigmented ( genotypically mutant ) PR cells display a degenerative phenotype , whereas virtually all pigmented ( genotypically wt ) PR cells are normal . χ2 ‐ test : 82 . 38 ; P0 . 001 . Total PR N = 310 ( pigmented ) and 327 ( not pigmented ) .

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

(C) Degeneration in mosaic ommatidia in ftfd clones. At this stage of degeneration approximately half of the non‐pigmented (genotypically mutant) PR cells display a degenerative phenotype, whereas virtually all pigmented (genotypically wt) PR cells are normal. χ2‐test: 82.38; P0.001. Total PR N=310 (pigmented) and 327 (not pigmented).


( D ) Tangential eye sections through MARCM clone mutants for ftfd that express either no transgene or an RNAi construct against yki ( UAS - ykiIR ) with Tub - Gal4 and aged 14 days at 29 ° C . Because the transgene UAS - ykiIR is on chromosomal arm 2 L , the same as ft , clones generated with this system carry two copies of UAS - ykiIR and therefore downregulate yki very effectively . No other cell outside the clones expresses the construct and is therefore wt for yki . Mask panel is presented on the right of each section . A quantification of photoreceptor numbers ( far right ) displays a very significant increase in the number of wt ommatidia ( N = 210 and 219 from four eyes ) . * * * P0 . 001 in two ‐ tailed t ‐ test . If all classes of ommatidia are considered , χ2 ‐ test = 232 . 30 ; P0 . 001 for 3 degrees of freedom .

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

(D) Tangential eye sections through MARCM clone mutants for ftfd that express either no transgene or an RNAi construct against yki (UAS-ykiIR) with Tub-Gal4 and aged 14 days at 29°C. Because the transgene UAS-ykiIR is on chromosomal arm 2 L, the same as ft, clones generated with this system carry two copies of UAS-ykiIR and therefore downregulate yki very effectively. No other cell outside the clones expresses the construct and is therefore wt for yki. Mask panel is presented on the right of each section. A quantification of photoreceptor numbers (far right) displays a very significant increase in the number of wt ommatidia (N=210 and 219 from four eyes). ***P0.001 in two‐tailed t‐test. If all classes of ommatidia are considered, χ2‐test=232.30; P0.001 for 3 degrees of freedom.


( A ) Tangential eye sections through wtsX1 mutant fly eyes . In clones for the null wtsX1 allele , degeneration inside the clones is at severe stages at 14 days .

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(A) Tangential eye sections through wtsX1 mutant fly eyes. In clones for the null wtsX1 allele, degeneration inside the clones is at severe stages at 14 days.


( B ) Tangential eye sections through sav3 clones . In sav3 after enclosure many photoreceptors are intact but after 14 days at 29 ° C most sav mutant photoreceptors have degenerated .

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(B) Tangential eye sections through sav3 clones. In sav3 after enclosure many photoreceptors are intact but after 14 days at 29°C most sav mutant photoreceptors have degenerated.


( C ) Histograms showing the quantification of ommatidia with the full complement of photoreceptors in hpo , wts and sav mutant clones . The progression of loss of photoreceptors is evident and statistically significant in all cases . Mutants for hpoBF33 do not survive for 14 days at 29oC and corresponding sections are shown in Supplementary Figure 12 . N = 195 - 268 . * * * P0 . 001 , * * P0 . 01 ; * P0 . 05 in two ‐ tailed t ‐ test .

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

(C) Histograms showing the quantification of ommatidia with the full complement of photoreceptors in hpo, wts and sav mutant clones. The progression of loss of photoreceptors is evident and statistically significant in all cases. Mutants for hpoBF33 do not survive for 14 days at 29oC and corresponding sections are shown in Supplementary Figure 12. N=195-268. ***P0.001, **P0.01; *P0.05 in two‐tailed t‐test.


( A ) Regression analysis of the overgrowth versus the neurodegeneration observed in loss of function mutants ( left ) and overexpression of transgenes ( right ) . Overgrowth quantification is shown in Supplementary Figure 14 . For loss of function mutant , the neurodegeneration was quantified at 14 days in clones ( as shown in Figures 4 and 5 ) . For UAS ‐ overexpression transgenes neurodegeneration was quantified at 14 days when expressed with GMRGal4 ; UbiGal80ts . For CycD + Cdk4 , these specific data are missing and have not been plotted ; however , Supplementary Figure 13 shows that these mutants do not cause neurodegeneration in a different setup in which they are expressed during both development and adult life . In both cases the regression coefficient r2 is extremely low ( 0 . 21 and 0 . 03 ) , indicating an absence of correlation between the two processes .

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(A) Regression analysis of the overgrowth versus the neurodegeneration observed in loss of function mutants (left) and overexpression of transgenes (right). Overgrowth quantification is shown in Supplementary Figure 14. For loss of function mutant, the neurodegeneration was quantified at 14 days in clones (as shown in Figures 4 and 5). For UAS‐overexpression transgenes neurodegeneration was quantified at 14 days when expressed with GMRGal4; UbiGal80ts. For CycD+Cdk4, these specific data are missing and have not been plotted; however, Supplementary Figure 13 shows that these mutants do not cause neurodegeneration in a different setup in which they are expressed during both development and adult life. In both cases the regression coefficient r2 is extremely low (0.21 and 0.03), indicating an absence of correlation between the two processes.


( B ) Tangential eye sections through the eyes of flies expressing with GMR - Gal4 , Ubi - Gal80ts either UAS - EGFP or UAS - Yki or UAS - YkiS111A , S168A , S250A and aged 1 or 28 days . Arrows point at missing or degenerated photoreceptors . The UAS - YkiS111A , S168A , S250A is so effective that even the very low expression leaking out with this system is enough to affect development and generate mild eye roughness and overproliferation of lattice cells . Histograms showing the quantification of cell loss in eyes shown in A and also eyes from flies aged at the intermediate 14 ‐ day stage . Mild but significant degeneration is observed for both Yki forms , with respect to the negative control ; however , no statistical difference is observed between the two Yki proteins that have dramatically different effects on overgrowth . * * * P0 . 001 , * * P0 . 01 ; * P0 . 05 in two ‐ tailed t ‐ test .

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(B) Tangential eye sections through the eyes of flies expressing with GMR-Gal4, Ubi-Gal80ts either UAS-EGFP or UAS-Yki or UAS-YkiS111A,S168A,S250A and aged 1 or 28 days. Arrows point at missing or degenerated photoreceptors. The UAS-YkiS111A,S168A,S250A is so effective that even the very low expression leaking out with this system is enough to affect development and generate mild eye roughness and overproliferation of lattice cells. Histograms showing the quantification of cell loss in eyes shown in A and also eyes from flies aged at the intermediate 14‐day stage. Mild but significant degeneration is observed for both Yki forms, with respect to the negative control; however, no statistical difference is observed between the two Yki proteins that have dramatically different effects on overgrowth. ***P0.001, **P0.01; *P0.05 in two‐tailed t‐test.


Neurodegeneration by polyQ Atro partially requires dachs . Tangential eye sections through dGC13 clones ( marked by the absence of yellow pigment ) either in control flies or those expressing Atro75QN with the Rhodopsin1 driver and aged 28 days . Mask panels below each section exemplify clonal boundaries and PR no . for the ommatidia . Early signs of degeneration indicated by reduced complement of photoreceptors are detected specifically in the pigmented area ( arrowhead ) . At this stage , inside d mutant clones all ommatidia are unaffected and display a wt arrangement ( arrows ) . ( B ) Histograms showing the quantification of cell loss in eyes shown in ( A ) . A statistically significant rescue is obtained both for the number of wt ommatidia ( * P0 . 05 in two ‐ tailed t ‐ test ) and for all other categories of ommatidia ( χ2 = 44 . 76 ; P0 . 001 for 2 degrees of freedom ) .

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Neurodegeneration by polyQ Atro partially requires dachs. Tangential eye sections through dGC13 clones (marked by the absence of yellow pigment) either in control flies or those expressing Atro75QN with the Rhodopsin1 driver and aged 28 days. Mask panels below each section exemplify clonal boundaries and PR no. for the ommatidia. Early signs of degeneration indicated by reduced complement of photoreceptors are detected specifically in the pigmented area (arrowhead). At this stage, inside d mutant clones all ommatidia are unaffected and display a wt arrangement (arrows). (B) Histograms showing the quantification of cell loss in eyes shown in (A). A statistically significant rescue is obtained both for the number of wt ommatidia (*P0.05 in two‐tailed t‐test) and for all other categories of ommatidia (χ2=44.76; P0.001 for 2 degrees of freedom).


( A ) EM scan of a wt ( left ) and ftfd mutant ommatidium ( right ) of a ftfd clone after 14 days . Scale bar : 1 μm . High ‐ magnification panels ( far right , top to bottom ) display autophagosomes with undigested debris , damaged mitochondria and forming phagophores ( arrowheads ) , found in ftfd mutant cells . Scale bar : 0 . 2 μm for zoom ‐ in panels .

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(A) EM scan of a wt (left) and ftfd mutant ommatidium (right) of a ftfd clone after 14 days. Scale bar: 1 μm. High‐magnification panels (far right, top to bottom) display autophagosomes with undigested debris, damaged mitochondria and forming phagophores (arrowheads), found in ftfd mutant cells. Scale bar: 0.2 μm for zoom‐in panels.


( B ) Graphs of the quantification of autophagic vesicles ( AV ) per photoreceptor found in EM sections of 7 ‐ day ( left ) ‐ and 14 ‐ day ( right ) ‐ old ftfd clones . Significant accumulation of AV is found in mutant cells ( not pigmented ) with respect to genotypically wt ( pigmented ) cells . * * P0 . 01 and * * * P0 . 001 in one ‐ tailed t ‐ test . N = 12 pigmented cells versus 20 non ‐ pigmented cells for the 7 ‐ day graph and 13 pigmented cells versus 21 non ‐ pigmented cells for the 14 ‐ day graph .

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(B) Graphs of the quantification of autophagic vesicles (AV) per photoreceptor found in EM sections of 7‐day (left)‐ and 14‐day (right)‐old ftfd clones. Significant accumulation of AV is found in mutant cells (not pigmented) with respect to genotypically wt (pigmented) cells. **P0.01 and ***P0.001 in one‐tailed t‐test. N=12 pigmented cells versus 20 non‐pigmented cells for the 7‐day graph and 13 pigmented cells versus 21 non‐pigmented cells for the 14‐day graph.


( C ) Confocal pictures of whole ‐ mount retinae of a ftfd clone aged 7 days and expressing GFP ∷ Atg8a ubiquitously with Tub - Gal4 . Red is phalloidin marking rhabdomeres , green is GFP and the clone is marked by the absence of β ‐ gal staining ( blue ) . Small GFP ∷ Atg8a dots accumulate specifically inside ft mutant cells ( arrow ) .

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(C) Confocal pictures of whole‐mount retinae of a ftfd clone aged 7 days and expressing GFP∷Atg8a ubiquitously with Tub-Gal4. Red is phalloidin marking rhabdomeres, green is GFP and the clone is marked by the absence of β‐gal staining (blue). Small GFP∷Atg8a dots accumulate specifically inside ft mutant cells (arrow).


( D ) Confocal pictures of whole ‐ mount retinae of a ftfd clone aged 7 days ( left ) and 14 days ( right ) . Red is phalloidin marking rhabdomeres , blue is p62 and the clone is marked by the absence of GFP staining ( green ) . p62 starts to gather in small dots specifically inside ft mutant cells ( arrow ) and then accumulates massively ( arrow ) in the ft mutant clones as many cells degenerate .

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(D) Confocal pictures of whole‐mount retinae of a ftfd clone aged 7 days (left) and 14 days (right). Red is phalloidin marking rhabdomeres, blue is p62 and the clone is marked by the absence of GFP staining (green). p62 starts to gather in small dots specifically inside ft mutant cells (arrow) and then accumulates massively (arrow) in the ft mutant clones as many cells degenerate.


( A ) EM scan of a 7 ‐ day ‐ old sav3 mutant ommatidium and zoom ‐ in on autophagosomes containing undigested debris ( arrowhead ) . Scale bar : 1 μm for panel on the left and 0 . 2 μm for zoom ‐ in panel on the right .

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(A) EM scan of a 7‐day‐old sav3 mutant ommatidium and zoom‐in on autophagosomes containing undigested debris (arrowhead). Scale bar: 1 μm for panel on the left and 0.2 μm for zoom‐in panel on the right.


( B ) Graph of the quantification of autophagic vesicles ( AV ) per photoreceptor found in EM sections of 1 ‐ week ‐ old sav3 clones . Significant accumulation of AV is found in mutant cells . * * * P0 . 001 in one ‐ tailed t ‐ test . N = 45 pigmented cells versus 42 non ‐ pigmented cells .

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(B) Graph of the quantification of autophagic vesicles (AV) per photoreceptor found in EM sections of 1‐week‐old sav3 clones. Significant accumulation of AV is found in mutant cells. ***P0.001 in one‐tailed t‐test. N=45 pigmented cells versus 42 non‐pigmented cells.


A . Rab25 expression decreases nutrient withdrawal ( 24 h ) induced cell death in ovarian A2780 and IOSE29 cells measured using the Cell death ELISA plus assay ( left ) ; cells were cultured in complete media ( containing 5 % FBS ) , serum free media ( SF ) , complete media plus 2DG , serum and glucose free media ( SF - glu ) , or in amino acid , glucose and SF Earls buffer salt solution ( EBSS ) . ( a ) p < 0 . 01 versus control and ( b ) p < 0 . 05 versus pcDNA . ( Right ) Percentage of apoptotic cells ( sub - G0 population ) by flow cytometry in A2780 cells following glucose and serum withdrawal ( Glu / SF ) . ( a ) p < 0 . 001 versus A2780pcDNA control and ( b ) p < 0 . 05 A2780Rab25 versus A2780pcDNA control .

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A. Rab25 expression decreases nutrient withdrawal (24 h) induced cell death in ovarian A2780 and IOSE29 cells measured using the Cell death ELISA plus assay (left); cells were cultured in complete media (containing 5% FBS), serum free media (SF), complete media plus 2DG, serum and glucose free media (SF-glu), or in amino acid, glucose and SF Earls buffer salt solution (EBSS). (a) p < 0.01 versus control and (b) p < 0.05 versus pcDNA. (Right) Percentage of apoptotic cells (sub-G0 population) by flow cytometry in A2780 cells following glucose and serum withdrawal (Glu/SF). (a) p < 0.001 versus A2780pcDNA control and (b) p < 0.05 A2780Rab25 versus A2780pcDNA control.


B . Decreasing Rab25 expression increases sensitivity to nutrient stress induced cell death . Expression of Rab25 was down - regulated by siRNA or shRNA specific to Rab25 .

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B. Decreasing Rab25 expression increases sensitivity to nutrient stress induced cell death. Expression of Rab25 was down-regulated by siRNA or shRNA specific to Rab25.


C . Rab25 regulates autophagy activity in ovarian cancer cells under serum and glucose deprivation conditions . Western blot for the 16kD LC3 - II fragment , indicative of autophagy activity , in A2780 cells after 4 and 6 h of serum and glucose withdrawal .

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C. Rab25 regulates autophagy activity in ovarian cancer cells under serum and glucose deprivation conditions. Western blot for the 16kD LC3-II fragment, indicative of autophagy activity, in A2780 cells after 4 and 6 h of serum and glucose withdrawal.


D . Electron microscopy of autophagy in ovarian A2780 cells after 4 and 24 h of serum and glucose withdrawal ( upper panel , low magnification ) . High magnification images of boxed areas with arrowheads depicting autophagic vacuoles ( lower panel , inset ; N : nucleus ) . Average number of autophagosomes per cell was calculated by counting the number of autophagosomes in 16 individual cells from two - independent experiments .

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D. Electron microscopy of autophagy in ovarian A2780 cells after 4 and 24 h of serum and glucose withdrawal (upper panel, low magnification). High magnification images of boxed areas with arrowheads depicting autophagic vacuoles (lower panel, inset; N: nucleus). Average number of autophagosomes per cell was calculated by counting the number of autophagosomes in 16 individual cells from two-independent experiments.


E - H . Expression of Rab25 decreases glucose and serum deprivation induced signalling activation . Protein expression was measured by RPPA or WB analysis . E . RPPA detection of time - dependent activation of AMPK after nutrient withdrawal .

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E-H. Expression of Rab25 decreases glucose and serum deprivation induced signalling activation. Protein expression was measured by RPPA or WB analysis.E. RPPA detection of time-dependent activation of AMPK after nutrient withdrawal.


E - H . Expression of Rab25 decreases glucose and serum deprivation induced signalling activation . Protein expression was measured by RPPA or WB analysis . F . WB analysis of AMPK and acetyl - CoA carboxylase ( ACC ) phosphorylation in HEY ovarian cancer cells ( upper panel ) . WB of phospho - ACC levels in A2780 , IOSE80ht and SKOV3 ovarian cells after 1 h of nutrient withdrawal ( lower panel ) .

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E-H. Expression of Rab25 decreases glucose and serum deprivation induced signalling activation. Protein expression was measured by RPPA or WB analysis.F. WB analysis of AMPK and acetyl-CoA carboxylase (ACC) phosphorylation in HEY ovarian cancer cells (upper panel). WB of phospho-ACC levels in A2780, IOSE80ht and SKOV3 ovarian cells after 1 h of nutrient withdrawal (lower panel).


E - H . Expression of Rab25 decreases glucose and serum deprivation induced signalling activation . Protein expression was measured by RPPA or WB analysis . G . RPPA detection of phosphorylation of ACC after withdrawal of serum and glucose .

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E-H. Expression of Rab25 decreases glucose and serum deprivation induced signalling activation. Protein expression was measured by RPPA or WB analysis.G. RPPA detection of phosphorylation of ACC after withdrawal of serum and glucose.


E - H . Expression of Rab25 decreases glucose and serum deprivation induced signalling activation . Protein expression was measured by RPPA or WB analysis . H . Effect of Rab25 down - regulation on AMPK ( left panel ) and ACC ( right panel ) phosphorylation . Total cellular protein , isolated from A2780pcDNA and Rab25 expressing cells 72 h post - transfection with either non - target ( NT ) RNAi or Rab25 specific RNAi , was separated by polyacrylamide gel electrophoresis ( PAGE ) followed by WB analysis . ( a ) p < 0 . 001 versus NT RNAi control and ( b ) p < 0 . 001 , pcDNA versus Rab25 .

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E-H. Expression of Rab25 decreases glucose and serum deprivation induced signalling activation. Protein expression was measured by RPPA or WB analysis.H. Effect of Rab25 down-regulation on AMPK (left panel) and ACC (right panel) phosphorylation. Total cellular protein, isolated from A2780pcDNA and Rab25 expressing cells 72 h post-transfection with either non-target (NT) RNAi or Rab25 specific RNAi, was separated by polyacrylamide gel electrophoresis (PAGE) followed by WB analysis. (a) p < 0.001 versus NT RNAi control and (b) p < 0.001, pcDNA versus Rab25.


Expression of Rab25 increases endogenous ATP level in ovarian cancer cells . * p < 0 . 001 Rab25 versus pcDNA . The mean cellular concentration of ATP per mg of protein ( based on the standard curve generated using a known amount of ATP ) is 2 . 36 − 10 moles / mg for IOSE29htpcDNA cells , 6 . 21 − 10 moles / mg for IOSE29htRab25 cells , 8 . 14 − 11 moles / mg for IOSE80htpcDNA cells , 1 . 05 − 10 moles / mg for IOSE80htRab25 cells , 2 . 85 − 10 moles / mg for A2780pcDNA cells , 3 . 81 − 10 moles / mg for A2780Rab25 cells , 7 . 23 − 10 moles / mg for HEYpcDNA cells and 9 . 82 − 10 moles / mg for HEYRab25 cells .

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Expression of Rab25 increases endogenous ATP level in ovarian cancer cells. *p < 0.001 Rab25 versus pcDNA. The mean cellular concentration of ATP per mg of protein (based on the standard curve generated using a known amount of ATP) is 2.36−10 moles/mg for IOSE29htpcDNA cells, 6.21−10 moles/mg for IOSE29htRab25 cells, 8.14−11 moles/mg for IOSE80htpcDNA cells, 1.05−10 moles/mg for IOSE80htRab25 cells, 2.85−10 moles/mg for A2780pcDNA cells, 3.81−10 moles/mg for A2780Rab25 cells, 7.23−10 moles/mg for HEYpcDNA cells and 9.82−10 moles/mg for HEYRab25 cells.


Rab25 expression increases ATP synthesis and delays fall in total cellular ATP levels after glucose and serum withdrawal , p < 0 . 001 Rab25 versus control pcDNA .

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Rab25 expression increases ATP synthesis and delays fall in total cellular ATP levels after glucose and serum withdrawal, p < 0.001 Rab25 versus control pcDNA.


Down - regulation of Rab25 expression decreases ability to maintain ATP after glucose and serum withdrawal ( a ) p < 0 . 05 SF - Glu versus FBS and ( b ) p < 0 . 05 versus control .

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Down-regulation of Rab25 expression decreases ability to maintain ATP after glucose and serum withdrawal (a) p < 0.05 SF-Glu versus FBS and (b) p < 0.05 versus control.


Expression of Rab25 increases total cellular glycogen content while down - regulation of Rab25 expression decreases total cellular glycogen content ( µg of glycogen / mg protein ) . Cells were cultured in complete media for 24 h before glycogen measurement . ( a ) p < 0 . 01 Rab25 versus pcDNA , ( b ) p < 0 . 05 Rab25 RNAi versus NT RNAi and ( c ) p < 0 . 05 shRAb25 versus shRNA control .

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Expression of Rab25 increases total cellular glycogen content while down-regulation of Rab25 expression decreases total cellular glycogen content (µg of glycogen/mg protein). Cells were cultured in complete media for 24 h before glycogen measurement. (a) p < 0.01 Rab25 versus pcDNA, (b) p < 0.05 Rab25 RNAi versus NT RNAi and (c) p < 0.05 shRAb25 versus shRNA control.


Decrease in cellular glycogen content after glucose and serum withdrawal * p < 0 . 001 Rab25 versus pcDNA .

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Decrease in cellular glycogen content after glucose and serum withdrawal *p < 0.001 Rab25 versus pcDNA.


Effect of GPi ( Bay U6571 ) and oligomycin on ATP production . A2780pcDNA transfected and Rab25 expressing cells were pretreated with vehicle ( control ) , 30 µM of Bay U6571 ( GPi ) or 25 µg / ml of oligomycin for 2 h . Cells were then cultured in serum - and glucose free RPMI 1640 in the presence or absence of Bay U6571 or oligomycin for the indicated times . ( a ) p < 0 . 01 oligomycin time 0 versus control time 0 and ( b ) p < 0 . 01 versus time 0 at each treatment group .

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Effect of GPi (Bay U6571) and oligomycin on ATP production. A2780pcDNA transfected and Rab25 expressing cells were pretreated with vehicle (control), 30 µM of Bay U6571 (GPi) or 25 µg/ml of oligomycin for 2 h. Cells were then cultured in serum- and glucose free RPMI 1640 in the presence or absence of Bay U6571 or oligomycin for the indicated times. (a) p < 0.01 oligomycin time 0 versus control time 0 and (b) p < 0.01 versus time 0 at each treatment group.


Effect of Bay U6571 on cellular glycogen ( left panel ) and ATP ( right panel ) levels in HEY cells . ( a ) p < 0 . 01 versus pcDNA control time 0 at 5 % FBS and ( b ) p < 0 . 01 GPi versus control at each corresponding treatment .

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Effect of Bay U6571 on cellular glycogen (left panel) and ATP (right panel) levels in HEY cells. (a) p < 0.01 versus pcDNA control time 0 at 5% FBS and (b) p < 0.01 GPi versus control at each corresponding treatment.