D Quantification of the percentage of the colon surface occupied by tumours . Intestine samples were stained for β - Catenin , and tumour regions with nuclear β - Catenin localisation were quantified using NDP . view software . Two - tailed p - value of unpaired t - test is indicated ; ns , not significant ( p > 0 . 05 ) . Mean ± SD is shown . ( n = 6 biological replicates )

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D Quantification of the percentage of the colon surface occupied by tumours. Intestine samples were stained for β-Catenin, and tumour regions with nuclear β-Catenin localisation were quantified using NDP.view software. Two-tailed p-value of unpaired t-test is indicated; ns, not significant (p > 0.05). Mean ± SD is shown. (n=6 biological replicates)


E Example of IHC with β - Catenin staining of tumour - free regions with membrane β - catenin localisation ( left ) and tumour regions with nuclear β - catenin localisation ( right ) . Scale bars , 50μm .

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E Example of IHC with β-Catenin staining of tumour-free regions with membrane β-catenin localisation (left) and tumour regions with nuclear β-catenin localisation (right). Scale bars, 50μm.


A Genotyping confirms the loss of Cdk8 exon 2 in Cdk8 - / - and Cdk8 - / - / Cdk19 - / - organoids after 7 days of OH - tamoxifen treatment .

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A Genotyping confirms the loss of Cdk8 exon 2 in Cdk8 -/- and Cdk8 -/-/Cdk19 -/- organoids after 7 days of OH-tamoxifen treatment.


B WB of organoid samples after 7 days of OH - tamoxifen treatment ; β - actin was used as loading control .

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B WB of organoid samples after 7 days of OH-tamoxifen treatment; β-actin was used as loading control.


C Phase contrast images of organoids before and after 6 days of OH - tamoxifen treatment . Scale bars , 150μm . D Quantification of organoid size at day 6 , shown in ( C ) ( mean ± SEM are shown ) . P - value , one - way ANOVA ( Bonferroni test ) : ( * ) p ≤ 0 . 05 , ( * * * ) p ≤ 0 . 001 ; ns , not significant ( p > 0 . 05 ) . ( n = 47 biological replicates )

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C Phase contrast images of organoids before and after 6 days of OH-tamoxifen treatment. Scale bars, 150μm. D Quantification of organoid size at day 6, shown in (C) (mean ± SEM are shown). P-value, one-way ANOVA (Bonferroni test): (*) p ≤ 0.05, (***) p ≤ 0.001; ns, not significant (p > 0.05). (n=47 biological replicates)


E IHC staining of organoids ( day 7 of OH - tamoxifen treatment ) with Ki - 67 antibody . Scale bars , 100μm . F Quantification of Ki - 67 positive area ( % of the total area of the organoids ; mean ± SEM ) in the four different genotypes presented in ( E ) . Areas with positive Ki - 67 signal were detected and quantified using QuPath and ImageJ programs . Four technical replicates from 2 biological replicates of each genotype were analysed . Adjusted p - values of ordinary one - way ANOVA ( Bonferroni test ) are indicated : ( * * ) p - values ≤ 0 . 01 ; ( * ) p - values ≤ 0 . 05 ; ns , not significant ( p > 0 . 05 ) .

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E IHC staining of organoids (day 7 of OH-tamoxifen treatment) with Ki-67 antibody. Scale bars, 100μm. F Quantification of Ki-67 positive area (% of the total area of the organoids; mean ± SEM) in the four different genotypes presented in (E). Areas with positive Ki-67 signal were detected and quantified using QuPath and ImageJ programs. Four technical replicates from 2 biological replicates of each genotype were analysed. Adjusted p-values of ordinary one-way ANOVA (Bonferroni test) are indicated: (**) p-values ≤ 0.01; (*) p-values ≤ 0.05; ns, not significant (p > 0.05).


C Left , IHC staining of WT and Cdk8 - / - / Cdk19 - / - organoids with Olfm4 ( stem cells ) , Lysozyme ( Paneth cells ) , Muc2 ( goblet cells ) , and Dclk1 ( tuft cells ) antibodies . Scale bars , 50μm ( Olfm4 , Muc2 , Dclk1 ) and 25μm ( Lysozyme ) . Right , quantification of Olfm4 , Lysozyme and Muc2 positive area ( % of the total organoid area as indicated by hematoxylin staining ; mean ± SEM ) , or the number of Dclk1 positive cells per organoid . P - value , unpaired two - tailed t - test : ( * * * ) p - value ≤ 0 . 001 ; ns , not significant ( p > 0 . 05 ) . 20 different organoids from 2 biological replicates of each genotype were used for the analysis .

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C Left, IHC staining of WT and Cdk8 -/-/Cdk19 -/- organoids with Olfm4 (stem cells), Lysozyme (Paneth cells), Muc2 (goblet cells), and Dclk1 (tuft cells) antibodies. Scale bars, 50μm (Olfm4, Muc2, Dclk1) and 25μm (Lysozyme). Right, quantification of Olfm4, Lysozyme and Muc2 positive area (% of the total organoid area as indicated by hematoxylin staining; mean ± SEM), or the number of Dclk1 positive cells per organoid. P-value, unpaired two-tailed t-test: (***) p-value ≤ 0.001; ns, not significant (p > 0.05). 20 different organoids from 2 biological replicates of each genotype were used for the analysis.


D WB of indicated proteins extracted from Wt and Cdk8 - / - / Cdk19 - / - organoids . Included are CFTR positive and negative control cell line samples obtained from the Cystic Fibrosis Foundation ( CFF ) . Amidoblack was used as loading control .

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D WB of indicated proteins extracted from Wt and Cdk8 -/-/Cdk19 -/- organoids. Included are CFTR positive and negative control cell line samples obtained from the Cystic Fibrosis Foundation (CFF). Amidoblack was used as loading control.


A Histological PAS staining of organoids treated for 7 days with OH - tamoxifen . Scale bar , 50μm . B Quantification of PAS signal ( % of total organoid area ; mean ± SD are shown ) in the four different genotypes presented in ( A ) . Areas containing positive PAS staining were detected and quantified using QuPath and ImageJ programs . Adjusted p - values of ordinary one - way Anova followed by Tukey ' s multiple comparison test are indicated : ( * * * ) p - value ≤ 0 . 001 ; ns : not significant ( p > 0 . 05 ) . Four technical replicates from 2 biological replicates of each genotype were analysed .

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A Histological PAS staining of organoids treated for 7 days with OH-tamoxifen. Scale bar, 50μm. B Quantification of PAS signal (% of total organoid area; mean ± SD are shown) in the four different genotypes presented in (A). Areas containing positive PAS staining were detected and quantified using QuPath and ImageJ programs. Adjusted p-values of ordinary one-way Anova followed by Tukey's multiple comparison test are indicated: (***) p-value ≤ 0.001; ns: not significant (p > 0.05). Four technical replicates from 2 biological replicates of each genotype were analysed.


C Representative phase contrast images of organoids at the indicated time points after 7 days of OH - tamoxifen treatment are shown . Scale bar , 100 μm . D Quantification of the time needed for mucus release ( observed as a dark staining in the center of the organoid ; mean ± SD are shown ) . Adjusted p - values of ordinary one - way Anova followed by Tukey ' s multiple comparison test are indicated : ( * * * ) p - value ≤ 0 . 001 ; ns : not significant ( p > 0 . 05 ) ; ( n = 17 for Wt , 11 for Cdk8 - / - , 18 for Cdk19 - / - , and 12 for Cdk8 - / - / Cdk19 - / - , all biological replicates ) .

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C Representative phase contrast images of organoids at the indicated time points after 7 days of OH-tamoxifen treatment are shown. Scale bar, 100 μm. D Quantification of the time needed for mucus release (observed as a dark staining in the center of the organoid; mean ± SD are shown). Adjusted p-values of ordinary one-way Anova followed by Tukey's multiple comparison test are indicated: (***) p-value ≤ 0.001; ns: not significant (p > 0.05); (n= 17 for Wt, 11 for Cdk8 -/-, 18 for Cdk19 -/-, and 12 for Cdk8 -/-/ Cdk19 -/- , all biological replicates).


E Fluorescence confocal microscopy images of Calcein green - labeled WT and Cdk8 - / - / Cdk19 - / - organoids treated with forskolin . Scale bars , 100 μm . F Quantification of forskolin - induced swelling in WT organoids treated for 1hour or 24 hours with 0 . 1μM , 1μM or 10μM Senexin B ( SenB ) , as indicated , or double KO organoids ; DMSO vehicle was used as control . The surface area of individual organoids at different time points relative to the area at t = 0 ( 100 % ) was measured ( mean ± SD , n = 8 biological replicates ) . Linear regression lines are shown .

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E Fluorescence confocal microscopy images of Calcein green-labeled WT and Cdk8-/-/Cdk19-/- organoids treated with forskolin. Scale bars, 100 μm. F Quantification of forskolin-induced swelling in WT organoids treated for 1hour or 24 hours with 0.1μM, 1μM or 10μM Senexin B (SenB), as indicated, or double KO organoids; DMSO vehicle was used as control. The surface area of individual organoids at different time points relative to the area at t = 0 (100%) was measured (mean ± SD, n=8 biological replicates). Linear regression lines are shown.


H WB of indicated proteins extracted from Senexin B - treated ( 10μM , 2h and 24h ) organoids ( left ) , and WT and Cdk8 - / - / Cdk19 - / - organoids ( right ) . Amidoblack was used as loading control .

H WB of indicated proteins extracted from Senexin B-treated (10μM, 2h and 24h) organoids (left), and WT and Cdk8 -/-/ Cdk19 -/- organoids (right). Amidoblack was used as loading control.


A WB of indicated proteins extracted from intestinal epithelium of control mice or mice treated with Senexin 631 ( Snx 631 ) for 4 or 11 days .

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A WB of indicated proteins extracted from intestinal epithelium of control mice or mice treated with Senexin 631 (Snx 631) for 4 or 11 days.


B Left , immunohistochemical PAS and hematoxylin staining of mouse small intestines collected at 4 and 11 days of Senexin 631 ( Snx 631 ) treatment ; scale bars 50μm . Right , quantification of PAS - and hematoxilin ( Hema ) - positive area ( mean ± SEM ) , using QuPath and ImageJ software . Adjusted p - values of unpaired t - test are shown : ( * ) p < 0 . 05 ; ns , not significant ( p > 0 . 05 ) . 8 mice ( 4 treated with Senexin 631 and and 4 control ) were used in this quantification .

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B Left, immunohistochemical PAS and hematoxylin staining of mouse small intestines collected at 4 and 11 days of Senexin 631 (Snx 631) treatment; scale bars 50μm. Right, quantification of PAS- and hematoxilin (Hema)-positive area (mean ± SEM), using QuPath and ImageJ software. Adjusted p-values of unpaired t-test are shown: (*) p < 0.05; ns, not significant (p > 0.05). 8 mice (4 treated with Senexin 631 and and 4 control) were used in this quantification.


A , Representative immunofluorescence staining of normal , tumor , and residual 3D organoids grown from primary mammary cells of a preclinical mouse model of breast cancer ( Methods ) . Similar morphology of normal and residual organoids shown on the left with polarity markers ITGA6 ( red ) , ZO - 1 ( green ) , GM - 130 ( magenta ) ; DAPI ( blue ) . Right : human MYC oncoprotein ( green ) is expressed only in tumor cells , CDH1 ( red ) . Scale bar : 25 μm . Data information : Number of replicates corresponds to individual mice used to derive organoids .

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A, Representative immunofluorescence staining of normal, tumor, and residual 3D organoids grown from primary mammary cells of a preclinical mouse model of breast cancer (Methods). Similar morphology of normal and residual organoids shown on the left with polarity markers ITGA6 (red), ZO-1 (green), GM-130 (magenta); DAPI (blue). Right: human MYC oncoprotein (green) is expressed only in tumor cells, CDH1 (red). Scale bar: 25 μm. Data information: Number of replicates corresponds to individual mice used to derive organoids.


, Selective secreted metabolites with significant change linked to urea cycle components from healthy ( n = 3 ; blue ) and regressed ( n = 3 ; green ) mammary glands . Values represent metabolite abundance levels as quantified by the area under the curve ( AUC ) of the marker fragment ions / transitions for each metabolite . Values are plotted on the log2 scale . Statistics were calculated using the limma package n R with the significance threshold corresponding to a Benjamini - Hochberg adjusted p - value ≤ 0 . 05 ( residual compared to normal ) . ata information : umbers of replicates correspond to individual mice or humans . ox plots : midline , median ; box , 25 - 75th percentile ; whisker , minimum to maximum . umbers marking comparisons ( gray lines ) show p values ( corresponding statistical tests are described in individual panel legends ) .

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, Selective secreted metabolites with significant change linked to urea cycle components from healthy (n=3; blue) and regressed (n=3; green) mammary glands. Values represent metabolite abundance levels as quantified by the area under the curve (AUC) of the marker fragment ions/transitions for each metabolite. Values are plotted on the log2 scale. Statistics were calculated using the limma package n R with the significance threshold corresponding to a Benjamini-Hochberg adjusted p-value ≤ 0.05 (residual compared to normal). ata information: umbers of replicates correspond to individual mice or humans. ox plots: midline, median; box, 25-75th percentile; whisker, minimum to maximum. umbers marking comparisons (gray lines) show p values (corresponding statistical tests are described in individual panel legends).


C , Left , Quantification of cells expressing ARG1 ( top ) , an enzyme converting arginine to urea and ornithine , and intensity of ARG1 ( bottom ) in normal cells from healthy ( n = 5 , 2921 cells analyzed ; blue ) and residual cells from regressed ( n = 5 , 2241 cells analyzed ; green ) mammary gland tissue sections . Statistical differences were calculated with the Mann - Whitney U test ( Wilcoxon rank - sum test ) . Right , Representative images of immunofluorescence staining in normal ( top ) and residual ( bottom ) duct stained for ARG1 ( green ) , CDH1 ( red ) , and DAPI ( blue ) . Scale bar : 20 μm . Data information : Numbers of replicates correspond to individual mice or humans . Box plots : midline , median ; box , 25 - 75th percentile ; whisker , minimum to maximum . Numbers marking comparisons ( gray lines ) show p values ( corresponding statistical tests are described in individual panel legends ) .

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C, Left, Quantification of cells expressing ARG1 (top), an enzyme converting arginine to urea and ornithine, and intensity of ARG1 (bottom) in normal cells from healthy (n=5, 2921 cells analyzed; blue) and residual cells from regressed (n=5, 2241 cells analyzed; green) mammary gland tissue sections. Statistical differences were calculated with the Mann-Whitney U test (Wilcoxon rank-sum test). Right, Representative images of immunofluorescence staining in normal (top) and residual (bottom) duct stained for ARG1 (green), CDH1 (red), and DAPI (blue). Scale bar: 20 μm. Data information: Numbers of replicates correspond to individual mice or humans. Box plots: midline, median; box, 25-75th percentile; whisker, minimum to maximum. Numbers marking comparisons (gray lines) show p values (corresponding statistical tests are described in individual panel legends).


D , Nitric oxide synthase ( NOS ) activity , an enzyme involved in arginine metabolism , in healthy ( n = 3 ; blue ) and in residual ( n = 3 ; green ) mouse mammary glands . The difference is statistically significant by unpaired two - samples t - test . E , Fractional labeling of lactate after cultivation of isolated regressed ( n = 3 ; green ) and healthy ( n = 3 ; blue ) mouse mammary glands in cell growth medium supplemented with [ U - 13C ] glucose for 8 h ( Methods ) . The three - carbon labeled ( 13C ) isotopologue ( M + 3 ) of lactate is depicted and shows enrichment in the residual cells of the regressed mammary glands . The difference is statistically significant by unpaired two - samples t - test . Data information : Numbers of replicates correspond to individual mice or humans . Numbers marking comparisons ( gray lines ) show p values ( corresponding statistical tests are described in individual panel legends ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Nitric oxide synthase (NOS) activity, an enzyme involved in arginine metabolism, in healthy (n=3; blue) and in residual (n=3; green) mouse mammary glands. The difference is statistically significant by unpaired two-samples t-test. E, Fractional labeling of lactate after cultivation of isolated regressed (n=3; green) and healthy (n=3; blue) mouse mammary glands in cell growth medium supplemented with [U-13C] glucose for 8 h (Methods). The three-carbon labeled (13C) isotopologue (M+3) of lactate is depicted and shows enrichment in the residual cells of the regressed mammary glands. The difference is statistically significant by unpaired two-samples t-test. Data information: Numbers of replicates correspond to individual mice or humans. Numbers marking comparisons (gray lines) show p values (corresponding statistical tests are described in individual panel legends).


A , ( Top ) Experimental design and ( bottom ) quantification of cell death ( Methods ) of normal , tumor , and residual cells after 72 - h treatment with 3 - BP at the indicated doses ( n = 5 mice per condition ) . Two - way ANOVA with Tukey ' s HSD multiple comparison testing was utilized to calculate statistical significance . Dox , doxycycline . Data information : Box plots : midline , median ; box , 25 - 75th percentile ; whiskers , minimum to maximum . Number of replicates corresponds to individual mice used to derive organoids . Numbers marking comparisons ( gray lines ) show p values ( corresponding statistical tests are described in individual panel legends ) .

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A, (Top) Experimental design and (bottom) quantification of cell death (Methods) of normal, tumor, and residual cells after 72-h treatment with 3-BP at the indicated doses (n=5 mice per condition). Two-way ANOVA with Tukey's HSD multiple comparison testing was utilized to calculate statistical significance. Dox, doxycycline. Data information: Box plots: midline, median; box, 25-75th percentile; whiskers , minimum to maximum. Number of replicates corresponds to individual mice used to derive organoids. Numbers marking comparisons (gray lines) show p values (corresponding statistical tests are described in individual panel legends).


B , Representative bright - field images of normal ( left ) , tumor ( middle ) , and residual ( right ) organoids , treated with vehicle ( top ) or with 50 μM 3 - BP ( bottom ) . Scale bar , 100 μm .

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B, Representative bright-field images of normal (left), tumor (middle), and residual (right) organoids, treated with vehicle (top) or with 50 μM 3-BP (bottom). Scale bar, 100 μm.


C , ( Top ) Experimental design and ( bottom ) cell - death quantification of passaged residual and normal cells after 48 h of treatment with 3 - BP ( n = 5 mice per condition ) . Statistics were calculated with multiple t - tests . Data information : Box plots : midline , median ; box , 25 - 75th percentile ; whiskers minimum to maximum . Number of replicates corresponds to individual mice used to derive organoids . Numbers marking comparisons ( gray lines ) show p values ( corresponding statistical tests are described in individual panel legends ) .

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C, (Top) Experimental design and (bottom) cell-death quantification of passaged residual and normal cells after 48 h of treatment with 3-BP (n=5 mice per condition). Statistics were calculated with multiple t-tests. Data information: Box plots: midline, median; box, 25-75th percentile; whiskers minimum to maximum. Number of replicates corresponds to individual mice used to derive organoids. Numbers marking comparisons (gray lines) show p values (corresponding statistical tests are described in individual panel legends).


D , ( Main ) Extracellular glucose abundance changes upon treatment with 3 - BP in all three populations ( n = 4 mice per condition ) . Values represent the ratio of glucose abundance in 3 - BP - treated versus untreated cells . Statistics were calculated using the limma package in R with the significance threshold corresponding to a Benjamini - Hochberg adjusted p - value ≤ 0 . 05 ( residual compared to normal ) . ( Insert ) Fractional labeling of lactate in untreated and 3 - BP treated normal ( n = 2 ) and residual ( n = 2 ) cells following cultivation in growth medium supplemented with [ U - 13C ] glucose . The three - carbon labeled ( 13C ) isotopologue ( M + 3 ) of lactate is depicted . Statistics were calculated with unpaired two - samples t - tests . Data information : , Box plots : midline , median ; box , 25 - 75th percentile minimum to maximum . Number of replicates corresponds to individual mice used to derive organoids . Numbers marking comparisons ( gray lines ) show p values ( corresponding statistical tests are described in individual panel legends ) .

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D, (Main) Extracellular glucose abundance changes upon treatment with 3-BP in all three populations (n=4 mice per condition). Values represent the ratio of glucose abundance in 3-BP-treated versus untreated cells. Statistics were calculated using the limma package in R with the significance threshold corresponding to a Benjamini-Hochberg adjusted p-value ≤ 0.05 (residual compared to normal). (Insert) Fractional labeling of lactate in untreated and 3-BP treated normal (n=2) and residual (n=2) cells following cultivation in growth medium supplemented with [U-13C] glucose. The three-carbon labeled (13C) isotopologue (M+3) of lactate is depicted. Statistics were calculated with unpaired two-samples t-tests. Data information: , Box plots: midline, median; box, 25-75th percentile minimum to maximum. Number of replicates corresponds to individual mice used to derive organoids. Numbers marking comparisons (gray lines) show p values (corresponding statistical tests are described in individual panel legends).


( B ) GST , GST - MCM2N ( human ) and GST - Spt5N ( yeast ) were mixed as indicated with yeast histones H3 - H4 , in the presence of glutathione agarose beads ( see STAR method for further details ) . The input and pulldown samples were analysed by SDS - PAGE before staining with colloidal Coomassie blue . The asterisks denote truncations of GST - Spt5N . ( C ) Similar experiment to ( B ) but with histones H2A - H2B . ( D ) Similar experiment to ( B ) to compare histone binding by GST - MCM2N and GST - MCM2N - 2A ( with Y81A Y90A mutations of conserved residues ) .

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(B) GST, GST-MCM2N (human) and GST-Spt5N (yeast) were mixed as indicated with yeast histones H3-H4, in the presence of glutathione agarose beads (see STAR method for further details). The input and pulldown samples were analysed by SDS-PAGE before staining with colloidal Coomassie blue. The asterisks denote truncations of GST-Spt5N. (C) Similar experiment to (B) but with histones H2A-H2B. (D) Similar experiment to (B) to compare histone binding by GST-MCM2N and GST-MCM2N-2A (with Y81A Y90A mutations of conserved residues).


( H ) . Budding yeast cells expressing fusions of Spt5N and Spt5N - 3A to Protein A were grown in parallel with cells expressing the equivalent wild type and mutant versions of fission yeast Spt5N ( Sp Spt5N and Sp Spt5N - 3A - the latter contained the F146A , E150A and V153A mutations ) .

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(H). Budding yeast cells expressing fusions of Spt5N and Spt5N-3A to Protein A were grown in parallel with cells expressing the equivalent wild type and mutant versions of fission yeast Spt5N (Sp Spt5N and Sp Spt5N-3A - the latter contained the F146A, E150A and V153A mutations).


( C ) Yeast cells were generated that expressed Protein A - tagged versions of wild type Spt5 ( full length or the indicated truncations ) , or full length Spt5 - 3A , under the control of the GAL1 , 10 promoter . Cells were grown in medium containing galactose and the ProteinA - tagged Spt5 proteins were isolated from cell extracts on magnetic beads coated with IgG . Association of the tagged Spt5 proteins with the elongating form of Pol II was monitored by immunoblotting , using antibodies specific to the indicated phosphorylation sites in the C - Terminal Domain repeats of the Rpb1 subunit of Pol II .

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(C) Yeast cells were generated that expressed Protein A-tagged versions of wild type Spt5 (full length or the indicated truncations), or full length Spt5-3A, under the control of the GAL1,10 promoter. Cells were grown in medium containing galactose and the ProteinA-tagged Spt5 proteins were isolated from cell extracts on magnetic beads coated with IgG. Association of the tagged Spt5 proteins with the elongating form of Pol II was monitored by immunoblotting, using antibodies specific to the indicated phosphorylation sites in the C-Terminal Domain repeats of the Rpb1 subunit of Pol II.


( E ) Serial dilutions of control cells or spt5 - AID cells , expressing the indicated versions of Spt5 from the GAL1 , 10 promoter , were grown on the indicated medium .

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(E) Serial dilutions of control cells or spt5-AID cells, expressing the indicated versions of Spt5 from the GAL1,10 promoter, were grown on the indicated medium.


( B ) Immunoblot analysis at the indicated times for the samples described in ( A ) .

(B) Immunoblot analysis at the indicated times for the samples described in (A).


( C ) Cells from the end of the experiment described in ( A ) were fixed and processed for ChIP - Seq , using antibodies specific for phosphorylated Serine 5 in the C - Terminal Domain of the Pol II catalytic subunit Rpb1 ( CTD - Ser5P ) . For each of the three indicated strains , the histograms represent the normalized read density ( DNA sequence read count per million reads , or RPM ) , across a sample gene body .

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(C) Cells from the end of the experiment described in (A) were fixed and processed for ChIP-Seq, using antibodies specific for phosphorylated Serine 5 in the C-Terminal Domain of the Pol II catalytic subunit Rpb1 (CTD-Ser5P). For each of the three indicated strains, the histograms represent the normalized read density (DNA sequence read count per million reads, or RPM), across a sample gene body.


( A ) Samples from the experiment described in Figure 3A were also processed for MNase - Seq . For each of the three indicated strains , the histograms represent the normalized DNA sequence read count per million reads ( RPM ) , across a sample gene body .

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(A) Samples from the experiment described in Figure 3A were also processed for MNase-Seq. For each of the three indicated strains, the histograms represent the normalized DNA sequence read count per million reads (RPM), across a sample gene body.


( C ) Samples from the same experiment were further processed for ChIP - Seq using antibodies to histone H3K4me3 . For each of the three indicated strains , the normalised read count per million reads is presented across a sample gene body .

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(C) Samples from the same experiment were further processed for ChIP-Seq using antibodies to histone H3K4me3. For each of the three indicated strains, the normalised read count per million reads is presented across a sample gene body.


( F ) Volcano plot showing the expression levels of BRAFi - resistant genes ( identified in 501Mel by CRISPR - SAM screen ) in 12 human melanoma cell lines . The fold change corresponds to the ratio of expression levels found in resistant and sensitive cell lines . In red : selected genes . EGFR is considered as positive control and SMAD3 , BIRC3 and SLC9A5 ; selected as favorite genes for the next steps . SMAD3 , BIRC3 , SLC9A5 and AFAP1 are BRAFi - resistance genes and potent tumor - promoting genes ( BRAFi for

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(F) Volcano plot showing the expression levels of BRAFi-resistant genes (identified in 501Mel by CRISPR-SAM screen) in 12 human melanoma cell lines. The fold change corresponds to the ratio of expression levels found in resistant and sensitive cell lines. In red: selected genes. EGFR is considered as positive control and SMAD3, BIRC3 and SLC9A5; selected as favorite genes for the next steps. SMAD3, BIRC3, SLC9A5 and AFAP1 are BRAFi-resistance genes and potent tumor-promoting genes (BRAFi for PBV


( B ) Tumor growth curves from BRAFi - persister cells ( monitored during 5 months ) V for Vem - resistant cells and PB for PLX8394 - resistant cells .

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(B) Tumor growth curves from BRAFi-persister cells (monitored during 5 months) V for Vem-resistant cells and PB for PLX8394-resistant cells.


( G ) SMAD3 depletion ( siRNA # 1 & # 2 ) decreased cell density and increased BRAFi effect ( vemurafenib ) on BRAFi - resistant cells ( SKMel28R ) . CTR for non - targeting siRNA . DMSO for dimethylsulfoxide ( solvent of vemurafenib ; BRAFi ) . n = 3 biologically independent experiments . Each histogram represents the mean + s . d . ( H ) SMAD3 depletion ( siRNA # 1 & # 2 ) decreased cell density and increased BRAFi effect ( vemurafenib , BRAFi ) on BRAFi - resistant cells ( Me1402 ) . CTR for non - targeting siRNA . DMSO for dimethylsulfoxide ( solvent of vemurafenib ) . n = 3 biologically independent experiments . Each histogram represents the mean + s . d .

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(G) SMAD3 depletion (siRNA#1 & #2) decreased cell density and increased BRAFi effect (vemurafenib) on BRAFi-resistant cells (SKMel28R). CTR for non-targeting siRNA. DMSO for dimethylsulfoxide (solvent of vemurafenib; BRAFi). n=3 biologically independent experiments. Each histogram represents the mean + s.d. (H) SMAD3 depletion (siRNA#1 & #2) decreased cell density and increased BRAFi effect (vemurafenib, BRAFi) on BRAFi-resistant cells (Me1402). CTR for non-targeting siRNA. DMSO for dimethylsulfoxide (solvent of vemurafenib). n=3 biologically independent experiments. Each histogram represents the mean + s.d.


( L ) SMAD3 , Phospho - SMAD3 Ser423 / 425 and BIRC3 expression levels in melanoma cell lines . Four subtypes of melanoma cells ( U , undifferentiated ; NC , neural crest - like ; T , transitory ; M , melanocytic ) have been compared . 501Mel and M249 cells are melanocytic cells in contrast to dedifferentiated BRAFi - resistant cells ( R ) . Three couples of melanoma cell lines ( Sensitive ( S ) and ( R ) ) have been used to illustrate the SMAD3 and BIRC3 increases in BRAFi - resistant cell lines . HEK293 cells are used as control ( kidney ) . HSC70 serves as loading control for western - blot experiments . Each antibody has been evaluated on individual membrane ( explaining the three HSC70 , loading controls ) .

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(L) SMAD3, Phospho-SMAD3 Ser423/425 and BIRC3 expression levels in melanoma cell lines. Four subtypes of melanoma cells (U, undifferentiated; NC, neural crest-like; T, transitory; M, melanocytic) have been compared. 501Mel and M249 cells are melanocytic cells in contrast to dedifferentiated BRAFi-resistant cells (R). Three couples of melanoma cell lines (Sensitive (S) and (R)) have been used to illustrate the SMAD3 and BIRC3 increases in BRAFi-resistant cell lines. HEK293 cells are used as control (kidney). HSC70 serves as loading control for western-blot experiments. Each antibody has been evaluated on individual membrane (explaining the three HSC70, loading controls).


( O ) The chemical inhibition of SMAD3 by SIS3 ( SMAD3i ) improved current therapy effect ( BRAFi + MEKi ; Vem 5µM and Cobi 1µM ) on BRAFi - resistant cells ( SKMel28R , M229R & M238R ) . Cells have been

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(O) The chemical inhibition of SMAD3 by SIS3 (SMAD3i) improved current therapy effect (BRAFi+MEKi; Vem 5µM and Cobi 1µM) on BRAFi-resistant cells (SKMel28R, M229R & M238R). Cells have been treated


( B ) AhR activation by exogenous and endogenous ligands promotes SMAD3 induction . 501Mel cells AhR wild - type or knock - out have been exposed to exogenous and endogenous AhR ligands ; TCDD ( 5nM ) or ITE ( 10µM ) or the solvent ( DMSO ) during 10 days . n = 5 biologically independent experiments for AhR WT cells and n = 3 for AhR KO cells . Each histogram represents the mean + s . d . ( C ) AhR activation by exogenous and endogenous AhR ligands promotes TIPARP induction . 501Mel cells have been treated as described in B . n = 5 biologically independent experiments for AhR WT cells and n = 3 for AhR KO cells . Each histogram represents the mean + s . d . ;

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(B) AhR activation by exogenous and endogenous ligands promotes SMAD3 induction. 501Mel cells AhR wild-type or knock-out have been exposed to exogenous and endogenous AhR ligands; TCDD (5nM) or ITE (10µM) or the solvent (DMSO) during 10 days. n=5 biologically independent experiments for AhR WT cells and n=3 for AhR KO cells. Each histogram represents the mean + s.d. (C) AhR activation by exogenous and endogenous AhR ligands promotes TIPARP induction. 501Mel cells have been treated as described in B. n=5 biologically independent experiments for AhR WT cells and n=3 for AhR KO cells. Each histogram represents the mean + s.d.;


( E ) Loss of AhR reduces SMAD3 expression levels . SMAD3 expression has been investigated in SKMel28 cells AhR wild - type ( WT ) or knock - out ( KO ) . SKMel28R , have been obtained from SKMel28S by chronic exposure to non - lethal doses of BRAFi R for BRAFi - resistant SKMel28 cells and S for sensitive .

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(E) Loss of AhR reduces SMAD3 expression levels. SMAD3 expression has been investigated in SKMel28 cells AhR wild-type (WT) or knock-out (KO). SKMel28R, have been obtained from SKMel28S by chronic exposure to non-lethal doses of BRAFi R for BRAFi-resistant SKMel28 cells and S for sensitive.


( E ) Heatmap depicting mRNA levels of SMAD3 - signature in BRAF ( V600E ) non - treated melanoma patients ( dataset from TCGA ; SKCM , BRAF ( V600E ) mutated : n = 118 ) . Three pigmentation genes ( MITF , MLANA and TYR ) have been added to highlight the differentiation states of tumors . ~ 20 % of tumors are considered as dedifferentiated tumors ( red box ) with a high SMAD3 - signature .

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(E) Heatmap depicting mRNA levels of SMAD3-signature in BRAF(V600E) non-treated melanoma patients (dataset from TCGA; SKCM, BRAF(V600E) mutated: n=118). Three pigmentation genes (MITF, MLANA and TYR) have been added to highlight the differentiation states of tumors. ~20% of tumors are considered as dedifferentiated tumors (red box) with a high SMAD3-signature.


( H ) SMAD3 depletion decreases expression of SMAD3 - regulated genes . SMAD3 knock - down by siRNA decreased mRNA expression of BRAFi - resistance genes in SKMel28R and Me1402 . NRP1 mRNA was not detected in our experimental conditions .

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(H) SMAD3 depletion decreases expression of SMAD3-regulated genes. SMAD3 knock-down by siRNA decreased mRNA expression of BRAFi-resistance genes in SKMel28R and Me1402. NRP1 mRNA was not detected in our experimental conditions.


siRNA depletion of COPI increases autophagy . ( A ) siRNA depletion of β ′ - , β - , and α - COPI ( top ) and Sec23A and B ( bottom ) in 293 / GFP - LC3 cells for 48 h . Decreased protein levels of the corresponding subunits is shown by immunoblots .

siRNA depletion of COPI increases autophagy. (A) siRNA depletion of β′-, β-, and α-COPI (top) and Sec23A and B (bottom) in 293/GFP-LC3 cells for 48 h. Decreased protein levels of the corresponding subunits is shown by immunoblots.


( B ) Loss of β ′ - , α - , and β - COP but not subunits of COPII ( Sec 23A and B ) increase GFP - LC3 - II . In A and B , tubulin was the loading control .

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(B) Loss of β′-, α-, and β-COP but not subunits of COPII (Sec 23A and B) increase GFP-LC3-II. In A and B, tubulin was the loading control.


( C ) GFP - LC3 - positive vesicles were detectable in 293 / GFP - LC3 cells after siRNA depletion of β ′ - , α - , and β - COP but only basal levels of GFP - LC3 - positive vesicles are observed in control siRNA - depleted cells .

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(C) GFP-LC3-positive vesicles were detectable in 293/GFP-LC3 cells after siRNA depletion of β′-, α-, and β-COP but only basal levels of GFP-LC3-positive vesicles are observed in control siRNA-depleted cells.


Time course of COPI depletion shows that Golgi disperses before AVs are formed , but Golgi dispersal does not cause AV formation . ( A ) COPI subunits were depleted in 293 / GFP - LC3 cells and analyzed 24 , 36 , and 48 h after addition of siRNA . Immunoblotting for β ′ - and α - COP confirms loss of COP subunits .

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Time course of COPI depletion shows that Golgi disperses before AVs are formed, but Golgi dispersal does not cause AV formation. (A) COPI subunits were depleted in 293/GFP-LC3 cells and analyzed 24, 36, and 48 h after addition of siRNA. Immunoblotting for β′- and α-COP confirms loss of COP subunits.


( B ) GFP - LC3 - I and - II were monitored by immunoblots in parallel lysates using anti - GFP antibodies at the indicated times .

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(B) GFP-LC3-I and -II were monitored by immunoblots in parallel lysates using anti-GFP antibodies at the indicated times.


( C ) After 24 h siRNA treatment , depletion of β ′ - COP caused morphological changes and a reduction in perinuclear population of GM130 . Box indicates enlarged area .

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(C) After 24 h siRNA treatment, depletion of β′-COP caused morphological changes and a reduction in perinuclear population of GM130. Box indicates enlarged area.


( D ) Rab1a / b was depleted using siRNAs for 48 h . Lysates were probed with anti - Rab1 to confirm depletion .

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(D) Rab1a/b was depleted using siRNAs for 48 h. Lysates were probed with anti-Rab1 to confirm depletion.


( E ) β ′ - COP or Rab1a / b were depleted as in D , and analyzed by indirect immunofluorescence using anti - GM130 and GFP fluorescence . In β ′ - COP panel , the asterisk indicates cells that did not show an accumulation of GFP - LC3 - positive AVs .

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(E) β′-COP or Rab1a/b were depleted as in D, and analyzed by indirect immunofluorescence using anti-GM130 and GFP fluorescence. In β′-COP panel, the asterisk indicates cells that did not show an accumulation of GFP-LC3-positive AVs.


AV formation after siRNA depletion of COPI requires Atg5 and Atg7 . 293 / GFP - LC3 cells were incubated with siRNA for β ′ - , α - COP , or control siRNA alone or combined with siRNA against Atg5 or Atg7 for 48 h , then ( A ) lysed and analyzed by immunoblot for Atg5 , Atg7 , GFP - LC3 , and tubulin ( loading control ) ,

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AV formation after siRNA depletion of COPI requires Atg5 and Atg7. 293/GFP-LC3 cells were incubated with siRNA for β′-, α-COP, or control siRNA alone or combined with siRNA against Atg5 or Atg7 for 48 h, then (A) lysed and analyzed by immunoblot for Atg5, Atg7, GFP-LC3, and tubulin (loading control),


( B ) fixed for immunofluorescence analysis using anti - ERGIC - 53 antibodies , or GFP fluorescence . In A , the asterisk indicates nonspecific band ; in B and C , asterisk indicates cells that did not show a fragmented ERGIC - 53 .

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(B) fixed for immunofluorescence analysis using anti-ERGIC-53 antibodies, or GFP fluorescence. In A, the asterisk indicates nonspecific band; in B and C, asterisk indicates cells that did not show a fragmented ERGIC-53.


Correlative light - electron microscopy analysis of GFP - LC3 puncta demonstrates they are AVs . ( A ) Phase , ( B ) fluorescent , and ( C ) TEM of the same field of 293 / GFP - LC3 cells 48 h after siRNA depletion of α - COP . In B the image was inverted to allow better visualization of the GFP - LC3 - positive structures . ( D ) Higher magnification of cell shown with asterisk in A and C . Top region of cell of interest is enlarged in E to show the two AVs structures indicated by arrows in B . ( F and G ) Higher magnification of AVs shown by arrows in E .

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Correlative light-electron microscopy analysis of GFP-LC3 puncta demonstrates they are AVs. (A) Phase, (B) fluorescent, and (C) TEM of the same field of 293/GFP-LC3 cells 48 h after siRNA depletion of α-COP. In B the image was inverted to allow better visualization of the GFP-LC3-positive structures. (D) Higher magnification of cell shown with asterisk in A and C. Top region of cell of interest is enlarged in E to show the two AVs structures indicated by arrows in B. (F and G) Higher magnification of AVs shown by arrows in E.


Ub and p62 accumulate in α - and β ′ - COP - depleted 293 / GFP - LC3 cells . ( A ) 293 / GFP - LC3 cells were treated with control or β ′ - COP siRNA for 48 h , then fixed and labeled with anti - p62 and anti - ubiquitin antibodies for colocalization with GFP - LC3 .

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Ub and p62 accumulate in α- and β′-COP-depleted 293/GFP-LC3 cells. (A) 293/GFP-LC3 cells were treated with control or β′-COP siRNA for 48 h, then fixed and labeled with anti-p62 and anti-ubiquitin antibodies for colocalization with GFP-LC3.


( B ) HEK293 cells treated as in A and labeled with anti - ubiquitin and anti - p62 antibodies .

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(B) HEK293 cells treated as in A and labeled with anti-ubiquitin and anti-p62 antibodies.


( C ) Cryosections from β ′ - COP - depleted cells were labeled with anti - Ub antibodies followed by 10 - nm protein A - gold . AV , autophagosomes .

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(C) Cryosections from β′-COP-depleted cells were labeled with anti-Ub antibodies followed by 10-nm protein A-gold. AV, autophagosomes.


AVs in COPI - depleted cells are not degradative . ( A ) Long - lived protein degradation was assessed 72 h after transfection with control or β ′ - COP siRNA in 293 / GFP - LC3 cells . Cells were either starved of amino acids for 2 h ( St ) or incubated in fresh growth medium ( Fed ) for 2 h . The amount of protein degradation is presented as mean ± SEM .

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AVs in COPI-depleted cells are not degradative. (A) Long-lived protein degradation was assessed 72 h after transfection with control or β′-COP siRNA in 293/GFP-LC3 cells. Cells were either starved of amino acids for 2 h (St) or incubated in fresh growth medium (Fed) for 2 h. The amount of protein degradation is presented as mean ± SEM.


( B ) siControl - treated cells and β ′ - and α - COP were depleted for 48 h , then incubated with Lysotracker red ( 50 nM ) for 30 min before fixation . Merged image shows very little colocalization of GFP - LC3 AV - positive structures . Asterisk shows cell enlarged in

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(B) siControl-treated cells and β′- and α-COP were depleted for 48 h, then incubated with Lysotracker red (50 nM) for 30 min before fixation. Merged image shows very little colocalization of GFP-LC3 AV-positive structures. Asterisk shows cell enlarged in inset


( C ) After β ′ - and α - COP siRNA depletion , Magic Red - RR2 ( MR ) was used to detect active cathepsin B in cells which were fed or starved ( St ) for 2 h . The intensity of MR labeling was measured in live cells by flow cytometry .

(C) After β′- and α-COP siRNA depletion, Magic Red-RR2 (MR) was used to detect active cathepsin B in cells which were fed or starved (St) for 2 h. The intensity of MR labeling was measured in live cells by flow cytometry.


GFP - LC3 AVs are LAMP2 positive . ( A ) 293 / GFP - LC3 cells were incubated with control or β ′ - or α - COP siRNA for 48 h , fixed and labeled with anti - LAMP2 antibodies , followed by Alexa 555 anti - mouse antibodies . After COPI depletion many GFP - LC3 - positive AVs are positive for LAMP2 . Asterisk indicates cell enlarged in inset .

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GFP-LC3 AVs are LAMP2 positive. (A) 293/GFP-LC3 cells were incubated with control or β′- or α-COP siRNA for 48 h, fixed and labeled with anti-LAMP2 antibodies, followed by Alexa 555 anti-mouse antibodies. After COPI depletion many GFP-LC3-positive AVs are positive for LAMP2. Asterisk indicates cell enlarged in inset.


( B ) Cryosections of β ′ - COP - depleted cells labeled with anti - GFP ( left ) or anti - GFP and anti - LAMP2 ( right ) followed by protein A - gold as indicated . AV , autophagosomes , M , mitochondria .

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(B) Cryosections of β′-COP-depleted cells labeled with anti-GFP (left) or anti-GFP and anti-LAMP2 (right) followed by protein A-gold as indicated. AV, autophagosomes, M, mitochondria.


GFP - LC3 - positive AVs colocalize TGN46 and early endocytotic markers after COPI depletion . ( A ) 293 / GFP - LC3 cells were incubated with siRNA for β ′ - COP or control siRNA ( Cont ) for 48 h , processed for immunofluorescence , and labeled with antibodies for TGN46 .

GFP-LC3-positive AVs colocalize TGN46 and early endocytotic markers after COPI depletion. (A) 293/GFP-LC3 cells were incubated with siRNA for β′-COP or control siRNA (Cont) for 48 h, processed for immunofluorescence, and labeled with antibodies for TGN46.


( B ) siRNA - treated cells were fixed and labeled , or starved for 2 h ( St ) , and labeled with antibodies to EEA1 . (

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(B) siRNA-treated cells were fixed and labeled, or starved for 2 h (St), and labeled with antibodies to EEA1. (


( C ) Control - starved HEK293 cells were labeled for endogenous LC3 and EEA1 by double labeling after methanol fixation .

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(C) Control-starved HEK293 cells were labeled for endogenous LC3 and EEA1 by double labeling after methanol fixation.


( D ) After siRNA treatment , rhodamine - labeled dextran was internalized for 20 min , followed by a 40 - min chase after which the 293 / GFP - LC3 cells were fixed and analyzed by confocal microscopy .

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(D) After siRNA treatment, rhodamine-labeled dextran was internalized for 20 min, followed by a 40-min chase after which the 293/GFP-LC3 cells were fixed and analyzed by confocal microscopy.


Transferrin and EGF trafficking is inhibited by COPI depletion . 293 / GFP - LC3 cells were treated with β ′ - COP siRNA or control siRNA . After 48 h cells were incubated with Alexa 555 transferrin ( A ) for 2 min , ( B ) 30 min , or ( C ) 30 min followed by a 30 - min chase , then washed and fixed and analyzed by confocal microscopy .

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Transferrin and EGF trafficking is inhibited by COPI depletion. 293/GFP-LC3 cells were treated with β′-COP siRNA or control siRNA. After 48 h cells were incubated with Alexa 555 transferrin (A) for 2 min, (B) 30 min, or (C) 30 min followed by a 30-min chase, then washed and fixed and analyzed by confocal microscopy.


( D and E ) 125I - EGF was added to HeLa cells after 48 h of siRNA depletion of β ′ - COP , or control siRNA for 10 min . The cells were then washed as described in the Materials and methods , and chased for 30 , 60 , 120 , and 180 min ( including 10 - min incubation ) . ( D ) The amount of 125I - EGF internalized was shown as a percentage of the total 125I - EGF taken up in 10 min .

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(D and E) 125I-EGF was added to HeLa cells after 48 h of siRNA depletion of β′-COP, or control siRNA for 10 min. The cells were then washed as described in the Materials and methods, and chased for 30, 60, 120, and 180 min (including 10-min incubation). (D) The amount of 125I-EGF internalized was shown as a percentage of the total 125I-EGF taken up in 10 min.


( E ) The amount of degraded 125I - EGF was determined as a percentage of the total radioactivity present after TCA precipitation over the total 125I - EGF internalized . The experiment was performed three times in duplicate and the data shown is the mean ± SEM . The significance was determined by Student ' s t test . * , P ≤ 0 . 05 .

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(E) The amount of degraded 125I-EGF was determined as a percentage of the total radioactivity present after TCA precipitation over the total 125I-EGF internalized. The experiment was performed three times in duplicate and the data shown is the mean ± SEM. The significance was determined by Student's t test. *, P ≤ 0.05.


a , b , THP1 cells were stimulated with rotenone ( 10 µM ) , TTFA ( 10 µM ) or antimycin ( 40 µg ml − 1 ) for 6 h and then stained with Mitotracker green and Mitotracker deep red ( a ) or MitoSOX ( b ) for 30 min and analysed by flow cytometry .

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a, b, THP1 cells were stimulated with rotenone (10 µM), TTFA (10 µM) or antimycin (40 µg ml−1) for 6 h and then stained with Mitotracker green and Mitotracker deep red (a) or MitoSOX (b) for 30 min and analysed by flow cytometry.


c , THP1 cells were stimulated for 6 h with the indicated amounts of rotenone , TTFA or antimycin . Supernatants ( SN ) and cell extracts ( input ) were analysed by western blotting as indicated .

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c, THP1 cells were stimulated for 6 h with the indicated amounts of rotenone, TTFA or antimycin. Supernatants (SN) and cell extracts (input) were analysed by western blotting as indicated.


d , LPS - primed bone - marrow - derived macrophages ( BMDMs ) from wild - type ( WT ) , Nlrp3 or Ipaf deficient mice were stimulated for 6 h with MSU ( 150 µg ml − 1 ) , rotenone ( 10 µM for THP1 cells and 40 µM for BMDMs ) or antimycin ( 40 µg ml − 1 for THP1 cells and 10 µg ml − 1 for BMDMs ) . The release of caspase - 1 ( western blot ) was then determined . Data shown are representative of three independent experiments .

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d, LPS-primed bone-marrow-derived macrophages (BMDMs) from wild-type (WT), Nlrp3 or Ipaf deficient mice were stimulated for 6 h with MSU (150 µg ml−1), rotenone (10 µM for THP1 cells and 40 µM for BMDMs) or antimycin (40 µg ml−1 for THP1 cells and 10 µg ml−1 for BMDMs). The release of caspase-1 (western blot) was then determined. Data shown are representative of three independent experiments.


a , BMDMs expressing GFP - LC3 were stimulated with rotenone ( 40 µM ) for 3 h and the co - localization of mitochondria and GFP - LC3 dots were analysed using confocal microscopy .

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a, BMDMs expressing GFP-LC3 were stimulated with rotenone (40 µM) for 3 h and the co-localization of mitochondria and GFP-LC3 dots were analysed using confocal microscopy.


b , THP1 cells stimulated with 3 - methyladenine ( 3 - MA , 10 mM ) for 24 h or THP1 cells stably expressing shRNA against beclin 1 or ATG5 were stained with Mitotracker green and Mitotracker deep red for 30 min , and analysed by flow cytometry .

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b, THP1 cells stimulated with 3-methyladenine (3-MA, 10 mM) for 24 h or THP1 cells stably expressing shRNA against beclin 1 or ATG5 were stained with Mitotracker green and Mitotracker deep red for 30 min, and analysed by flow cytometry.


c , LPS - primed BMDMs from wild - type , Nlrp3 − / − , Asc − / − or Ipaf − / − knockout mice were stimulated with MSU ( 150 µg ml − 1 ) or 3 - MA ( 10 mM ) for 6 h and the release of active caspase - 1 and IL - 1b was determined .

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c, LPS-primed BMDMs from wild-type, Nlrp3−/−, Asc−/− or Ipaf −/− knockout mice were stimulated with MSU (150 µg ml−1) or 3-MA (10 mM) for 6 h and the release of active caspase-1 and IL-1b was determined.


d , THP1 cells stably expressing shRNA against beclin 1 or ATG5 were incubated for 24 h and media supernatants ( SN ) and inputs were analysed by western blotting .

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d, THP1 cells stably expressing shRNA against beclin 1 or ATG5 were incubated for 24 h and media supernatants (SN) and inputs were analysed by western blotting.


e , THP1 cells stably expressing shRNA against beclin 1 or ATG5 were stimulated with MSU or nigericin for 6 h . Data shown are representative of three independent experiments .

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e, THP1 cells stably expressing shRNA against beclin 1 or ATG5 were stimulated with MSU or nigericin for 6 h. Data shown are representative of three independent experiments.


a - d , THP1 cells expressing Flag - NLRP3 ( a , b ) or Flag - ASC ( c , d ) were analysed for the co - localization of Flag - NLRP3 with the endoplasmic reticulum ( ER ) marker calreticulin or mitochondria ( Mitotracker ) using confocal microscopy .

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a-d, THP1 cells expressing Flag-NLRP3 (a, b) or Flag-ASC (c, d) were analysed for the co-localization of Flag-NLRP3 with the endoplasmic reticulum (ER) marker calreticulin or mitochondria (Mitotracker) using confocal microscopy.


b , THP1 cells expressing Flag - NLRP3 were analysed for the co - localization of Flag - NLRP3 with the endoplasmic reticulum ( ER ) marker calreticulin using confocal microscopy .

b, THP1 cells expressing Flag-NLRP3 were analysed for the co-localization of Flag-NLRP3 with the endoplasmic reticulum (ER) marker calreticulin using confocal microscopy.


e , THP1 cells were stimulated with MSU or nigericin and then fractionated . The cytosolic , ER , MAM and mitochondrial fractions were analysed by western blotting as indicated in the text .

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e, THP1 cells were stimulated with MSU or nigericin and then fractionated. The cytosolic, ER, MAM and mitochondrial fractions were analysed by western blotting as indicated in the text.


f , THP1 cells stably expressing shRNA against NLRP3 were stimulated with nigericin and subcellular fractions analysed as in e .

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f, THP1 cells stably expressing shRNA against NLRP3 were stimulated with nigericin and subcellular fractions analysed as in e.


g , TXNIP associates with mitochondria after NLRP3 inflammasome activation in a ROS - dependent manner . THP1 cells were stimulated with MSU and nigericin , and TXNIP localization was investigated in the presence or absence of the antioxidant APDC . Data shown are representative of two or three independent experiments . Scale bars : a - g , 20 µm .

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g, TXNIP associates with mitochondria after NLRP3 inflammasome activation in a ROS-dependent manner. THP1 cells were stimulated with MSU and nigericin, and TXNIP localization was investigated in the presence or absence of the antioxidant APDC. Data shown are representative of two or three independent experiments. Scale bars: a-g, 20 µm.


a , THP1 cells stably expressing shVDAC1 were stimulated with various NLRP inflammasome activators and then analysed for caspase - 1 activation and IL - 1b secretion .

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a, THP1 cells stably expressing shVDAC1 were stimulated with various NLRP inflammasome activators and then analysed for caspase-1 activation and IL-1b secretion.


b , THP1 cells expressing shRNA against VDAC2 were stimulated with MSU , R837 , silica , alum or nigericin and analysed for IL - 1b secretion .

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b, THP1 cells expressing shRNA against VDAC2 were stimulated with MSU, R837, silica, alum or nigericin and analysed for IL-1b secretion.


c , THP1 cells expressing shRNA against VDAC1 were stimulated with MSU and nigericin , and NLRP3 localization was analysed by confocal microscopy . Scale bars : 20 µm .

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c, THP1 cells expressing shRNA against VDAC1 were stimulated with MSU and nigericin, and NLRP3 localization was analysed by confocal microscopy. Scale bars: 20 µm.


C ) Violin and boxplots of Pol II density at enhancer regions ( n = 8 , 144 ) as a means of quantifying eRNA production . p - values were calculated using a two - sided Wilcoxon rank - sum test . Box plots consist of the median ( central line ) , the 25th and 75th percentiles ( box ) and the highest / lowest value within 1 . 5 * interquartile range of the box ( whiskers ) . Data information : n = 3 - 4 biological replicates per age group for tNET -

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C) Violin and boxplots of Pol II density at enhancer regions (n = 8,144) as a means of quantifying eRNA production. p-values were calculated using a two-sided Wilcoxon rank-sum test. Box plots consist of the median (central line), the 25th and 75th percentiles (box) and the highest/lowest value within 1.5 * interquartile range of the box (whiskers). Data information: n=3-4 biological replicates per age group for tNET-seq


D ) Western Blots of cellular fractionations of young and aged liver samples probed with the indicated antibodies .

D) Western Blots of cellular fractionations of young and aged liver samples probed with the indicated antibodies.


E ) Heatmaps and average intensity profiles of RNA Pol II and its S2 and S5 phosphorylated forms in young and aged animals . Data information : n = 2 for ChIP -

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E) Heatmaps and average intensity profiles of RNA Pol II and its S2 and S5 phosphorylated forms in young and aged animals. Data information: n=2 for ChIP-seq


F ) Heatmaps and average intensity profiles of SPT4 ChIP - seq signal at TSSs of tNET genes in young and aged animals . Data information : n = 2 for ChIP -

F) Heatmaps and average intensity profiles of SPT4 ChIP-seq signal at TSSs of tNET genes in young and aged animals. Data information: n=2 for ChIP-seq


A ) BW5147 reporter cells stably expressing human FcγR - ζ chain chimeras or BW5147 parental cells ( grey / dashed ) were stained with FcγR specific conjugated mAbs as indicated and measured for surface expression of FcγRs via flow cytometry . Bar graphs show means of three independent experiments performed in technical duplicates . Error bars = SD .

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A) BW5147 reporter cells stably expressing human FcγR-ζ chain chimeras or BW5147 parental cells (grey/dashed) were stained with FcγR specific conjugated mAbs as indicated and measured for surface expression of FcγRs via flow cytometry. Bar graphs show means of three independent experiments performed in technical duplicates. Error bars = SD.


C ) Immobilized IC , immobilized IgG and IgG opsonized cells represent qualitatively similar ligands for FcγRs . Response curves of human FcγRs activated by opsonized cells ( 293T cells stably expressing CD20 + Rituximab [ Rtx ] ) , immobilized IC ( rec . soluble CD20 + Rtx ) and immobilized IgG ( Rtx ) . sICs formed using monovalent antigen ( rec . soluble CD20 + Rtx ) do not activate human FcγRs . X - Axis shows sample concentration determined by antibody molarity . Y - Axis shows FcγR activation determined by reporter cell mouse IL - 2 production ( OD 450nm ) . Two independent experiments performed in technical duplicates . Error bars = SD .

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C) Immobilized IC, immobilized IgG and IgG opsonized cells represent qualitatively similar ligands for FcγRs. Response curves of human FcγRs activated by opsonized cells (293T cells stably expressing CD20 + Rituximab [Rtx]), immobilized IC (rec. soluble CD20 + Rtx) and immobilized IgG (Rtx). sICs formed using monovalent antigen (rec. soluble CD20 + Rtx) do not activate human FcγRs. X-Axis shows sample concentration determined by antibody molarity. Y-Axis shows FcγR activation determined by reporter cell mouse IL-2 production (OD 450nm). Two independent experiments performed in technical duplicates. Error bars = SD.


Ultra - pure antigen ( Ag , TNF - α ) mixed with therapy - grade mAb ( infliximab , Ifx ) was used to generate sICs . X - Axis : concentrations of stimulant expressed as molarity of either mAb or Ag monomer and IC ( expressed as mAb molarity ) at a mAb : Ag ratio of 1 : 2 . Soluble antigen or soluble antibody alone served as negative controls and were not sufficient to activate human FcγRs . Immobilized IgG ( Rtx ) or immobilized FcγR - specific mAbs served as positive controls . Two independent experiments performed in technical duplicates . Error bars = SD . Error bars smaller than symbols are not shown . Left panel : IL - 2 quantification 16 h after reporter cell activation . Background ( blank ) was subtracted ( dashed line ) . IL - 2 was measured via anti - IL - 2 ELISA ( A450nm ) and IL - 2 concentrations were calculated from an IL - 2 standard . Right panel : Reporter cell CD69 expression 4 h post trigger was measured using flow cytometry . MFI were normalized to untreated cells ( ctrl . ) and are presented as fold - change increase .

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Ultra-pure antigen (Ag, TNF-α) mixed with therapy-grade mAb (infliximab, Ifx) was used to generate sICs. X-Axis: concentrations of stimulant expressed as molarity of either mAb or Ag monomer and IC (expressed as mAb molarity) at a mAb:Ag ratio of 1:2. Soluble antigen or soluble antibody alone served as negative controls and were not sufficient to activate human FcγRs. Immobilized IgG (Rtx) or immobilized FcγR-specific mAbs served as positive controls. Two independent experiments performed in technical duplicates. Error bars = SD. Error bars smaller than symbols are not shown. Left panel: IL-2 quantification 16 h after reporter cell activation. Background (blank) was subtracted (dashed line). IL-2 was measured via anti-IL-2 ELISA (A450nm) and IL-2 concentrations were calculated from an IL-2 standard. Right panel: Reporter cell CD69 expression 4 h post trigger was measured using flow cytometry. MFI were normalized to untreated cells (ctrl.) and are presented as fold-change increase.


A ) Negatively selected primary NK cells purified from PBMCs of three healthy donors were tested for activation markers using flow cytometry . NK cells were incubated with immobilized IgG ( mepolizumab , Mpz ) , soluble IC ( Mpz : IL - 5 = 1 : 1 ) , soluble Mpz or soluble IL - 5 ( all at 200 nM , 106 cells ) . Incubation with PMA and Ionomycin ( Iono ) served as a positive control . Incubation with medium alone served as a negative control . Each dot represents one donor measured in technical duplicates . Error bars = SD . One - way ANOVA ( Tukey ) ; * p < 0 . 05 , * * * * p < 0 . 0001 .

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A) Negatively selected primary NK cells purified from PBMCs of three healthy donors were tested for activation markers using flow cytometry. NK cells were incubated with immobilized IgG (mepolizumab, Mpz), soluble IC (Mpz:IL-5 = 1:1), soluble Mpz or soluble IL-5 (all at 200 nM, 106 cells). Incubation with PMA and Ionomycin (Iono) served as a positive control. Incubation with medium alone served as a negative control. Each dot represents one donor measured in technical duplicates. Error bars = SD. One-way ANOVA (Tukey); *p<0.05, ****p<0.0001.


B ) Negatively selected primary neutrophils purified from whole blood of four healthy donors were tested for adhesion and activation markers using flow cytometry . Neutrophils were incubated with immobilized IgG ( Mpz ) , soluble IC ( Mpz : IL - 5 = 1 : 1 ) , soluble Mpz or soluble IL - 5 ( all at 200 nM , 2 * 105 cells ) . Incubation with PMA or immobilized rituximab served as positive controls . Incubation with medium served as a negative control . Immobilized FcγRII and FcγRIII specific mAbs served as functional controls . Mean florescence intensity ( MFI ) values at t = 30 minutes of incubation are presented as increase over t = 0 min . Each dot represents one donor measured in technical duplicates . Error bars = SD . Two - way ANOVA compared to medium ( Dunnett ) ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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B) Negatively selected primary neutrophils purified from whole blood of four healthy donors were tested for adhesion and activation markers using flow cytometry. Neutrophils were incubated with immobilized IgG (Mpz), soluble IC (Mpz:IL-5 = 1:1), soluble Mpz or soluble IL-5 (all at 200 nM, 2*105 cells). Incubation with PMA or immobilized rituximab served as positive controls. Incubation with medium served as a negative control. Immobilized FcγRII and FcγRIII specific mAbs served as functional controls. Mean florescence intensity (MFI) values at t=30 minutes of incubation are presented as increase over t=0 min. Each dot represents one donor measured in technical duplicates. Error bars = SD. Two-way ANOVA compared to medium (Dunnett); *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.


B ) sICs of different size were generated by cross - titration according to the AF4 measurement . Reporter cells were incubated with fixed amounts of either mAb ( infliximab , left ) or Ag ( rhTNF - α , right ) and titrated amounts of antigen or antibody , respectively . X - Axis shows titration of either antigen or antibody , respectively ( TNF - α calculated as monomer ) . Two independent experiments performed in technical duplicates . Error bars = SD .

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B) sICs of different size were generated by cross-titration according to the AF4 measurement. Reporter cells were incubated with fixed amounts of either mAb (infliximab, left) or Ag (rhTNF-α, right) and titrated amounts of antigen or antibody, respectively. X-Axis shows titration of either antigen or antibody, respectively (TNF-α calculated as monomer). Two independent experiments performed in technical duplicates. Error bars = SD.


C ) Purified primary NK cells from three different donors were incubated with cross - titrated sICs as in B . NK cells were measured for MIP - 1β expression ( % positivity ) . Incubation with PMA and Ionomycin served as a positive control . Incubation with medium alone served as a negative control . Each dot represents one experimental replicate , the dashed line represents the mean values .

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C) Purified primary NK cells from three different donors were incubated with cross-titrated sICs as in B. NK cells were measured for MIP-1β expression (% positivity). Incubation with PMA and Ionomycin served as a positive control. Incubation with medium alone served as a negative control. Each dot represents one experimental replicate, the dashed line represents the mean values.


A ) Experiments shown for an exemplary group of 8 SLE patients and two healthy individuals . Dose - dependent reactivity of FcγRs IIIA and IIB / C was observed only for SLE patient sera and not for sera from healthy individuals . Each titration represents one donor measured in technical duplicates .

A) Experiments shown for an exemplary group of 8 SLE patients and two healthy individuals. Dose-dependent reactivity of FcγRs IIIA and IIB/C was observed only for SLE patient sera and not for sera from healthy individuals. Each titration represents one donor measured in technical duplicates.


B ) Activation of FcγRs IIB / C and IIIA by patient serum is mediated by serum derived sICs . Patient serum samples were depleted of sICs by PEG precipitation and the supernatant ( SN ) was compared to untreated serum regarding FcγR activation ( left ) . Shown is one exemplary experiment performed in technical duplicates . Error bars indicate reproducibility as range . IgG concentration in the precipitate ( PC ) , supernatant ( SN ) or unfractionated serum respectively is shown in the bar graph ( right ) .

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B) Activation of FcγRs IIB/C and IIIA by patient serum is mediated by serum derived sICs. Patient serum samples were depleted of sICs by PEG precipitation and the supernatant (SN) was compared to untreated serum regarding FcγR activation (left). Shown is one exemplary experiment performed in technical duplicates. Error bars indicate reproducibility as range. IgG concentration in the precipitate (PC), supernatant (SN) or unfractionated serum respectively is shown in the bar graph (right).


C - D ) Correlation of FcγR activation data from A with ( C ) patient SLEDAI or ( D ) conventional SLE disease markers ( α - dsDNA levels indicated as IU / ml or C3d concentrations indicated as mg / L ) . Left panel : FcγRIIIA ; right panel : FcγRIIB / C . FcγR activation from a dose - response curve as in A was calculated as area under curve ( AUC ) for each SLE patient represented by individual dots . AUC from SLE patients are expressed as fold change compared to the healthy control mean ( n = 4 ) . SLE patients with α - dsDNA levels below 50 IU / ml and C3d values below 6 mg / L were excluded . One - tailed Spearman ' s . Dotted lines show 95 % confidence bands .

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C-D) Correlation of FcγR activation data from A with (C) patient SLEDAI or (D) conventional SLE disease markers (α-dsDNA levels indicated as IU/ml or C3d concentrations indicated as mg/L). Left panel: FcγRIIIA; right panel: FcγRIIB/C. FcγR activation from a dose-response curve as in A was calculated as area under curve (AUC) for each SLE patient represented by individual dots. AUC from SLE patients are expressed as fold change compared to the healthy control mean (n=4). SLE patients with α-dsDNA levels below 50 IU/ml and C3d values below 6 mg/L were excluded. One-tailed Spearman's. Dotted lines show 95% confidence bands.


A ) Reporter cells expressing mFcγRIII , mFcγRIV or parental BW5147 cells were incubated with titrated amounts of synthetic sICs generated from rhTNF - α and mouse - anti - hTNF - α at a 1 : 1 ratio by mass . One experiment performed in technical duplicates . Error bars indicate reproducibility as range .

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A) Reporter cells expressing mFcγRIII, mFcγRIV or parental BW5147 cells were incubated with titrated amounts of synthetic sICs generated from rhTNF-α and mouse-anti-hTNF-α at a 1:1 ratio by mass. One experiment performed in technical duplicates. Error bars indicate reproducibility as range.


B ) Titrations of three mouse sera per group ( C57BL / 6 , K / BxN or NZB / WF1 ) were incubated with mFcγR reporter cells and FcγR activation was assessed as described above . Sera from BL / 6 mice served as negative control . Each symbol represents mean values of one mouse measured in technical duplicates . Error bars = Mean with SD .

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B) Titrations of three mouse sera per group (C57BL/6, K/BxN or NZB/WF1) were incubated with mFcγR reporter cells and FcγR activation was assessed as described above. Sera from BL/6 mice served as negative control. Each symbol represents mean values of one mouse measured in technical duplicates. Error bars = Mean with SD.


D . Northern blot analysis of V . cholerae wild - type strain examined for the expression of VcdRP monitored over bacterial growth in the indicated media . The solid triangle represents the band corresponding to the full length primary vcdRP transcript , whereas the open triangles correspond to the different processed isoforms . Probing with 5S rRNA confirmed equal loading .

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D. Northern blot analysis of V. cholerae wild-type strain examined for the expression of VcdRP monitored over bacterial growth in the indicated media. The solid triangle represents the band corresponding to the full length primary vcdRP transcript, whereas the open triangles correspond to the different processed isoforms. Probing with 5S rRNA confirmed equal loading.


A . Northern blot analysis of the expression of VcdRP and Spot42 sRNA monitored at low cell density ( OD600 of 0 . 1 ) and high cell density ( OD600 of 2 . 0 ) , in the absence ( - ) or presence ( + ) of cAMP ( 5mM f . c . ) supplemented externally in LB medium . The solid triangle indicates the band corresponding to the full - length primary transcript . Probing with 5S rRNA confirmed equal loading .

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A. Northern blot analysis of the expression of VcdRP and Spot42 sRNA monitored at low cell density (OD600 of 0.1) and high cell density (OD600 of 2.0), in the absence (-) or presence (+) of cAMP (5mM f.c.) supplemented externally in LB medium. The solid triangle indicates the band corresponding to the full-length primary transcript. Probing with 5S rRNA confirmed equal loading.


B . Western blot analysis ( top ) of CTX levels detected in secreted protein fractions of V . cholerae ΔhapR cells grown under AKI conditions and carrying the indicated plasmids . Coomassie - stained SDS gel ( bottom ) confirmed equal loading of the protein fractions . The normalized band intensities are indicated for each expression plasmid relative to pCtrl , which was set to 100 % .

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B. Western blot analysis (top) of CTX levels detected in secreted protein fractions of V. cholerae ΔhapR cells grown under AKI conditions and carrying the indicated plasmids. Coomassie-stained SDS gel (bottom) confirmed equal loading of the protein fractions. The normalized band intensities are indicated for each expression plasmid relative to pCtrl, which was set to 100%.


I - L . Relative fluorescence intensities ( y - axis ) of E . coli strains harboring the gene - specific translational reporters as in ( A - D ) or the corresponding M2 * variant for each gene , combined with an empty control plasmid or vcdRP expression plasmids ( pVcdRP and pVcdRP M2 ) measured using plate reader ( I - K ) or by Western blot analysis ( L ) . The fluorescence of pCtrl ( for each reporter fusion ) was set to 1 .

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I-L. Relative fluorescence intensities (y-axis) of E. coli strains harboring the gene-specific translational reporters as in (A-D) or the corresponding M2* variant for each gene, combined with an empty control plasmid or vcdRP expression plasmids (pVcdRP and pVcdRP M2) measured using plate reader (I-K) or by Western blot analysis (L). The fluorescence of pCtrl (for each reporter fusion) was set to 1.


A . Immunoprecipitation of chromosomally tagged GltA : : HA combined with either an empty vector control ( pCtrl ) or SPA - tagged vcdP over - expression plasmid , grown in LB medium to exponential phase ( OD600 of 0 . 5 ) . The SPA epitope contains the 3 × FLAG and the calmodulin binding peptide sequences separated by a TEV protease cleavage site ( Zeghouf et al , 2004 ) . Therefore , protein samples corresponding to the total input and cell lysates before and after subjecting to immunoprecipitation with anti - Flag antibody were loaded on a SDS - PAGE gel . Western blotting with anti - HA and anti - Flag antibodies confirmed the interaction of GltA with VcdP in vivo . RNAP served as loading control . The solid triangles indicate the corresponding protein sizes .

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A. Immunoprecipitation of chromosomally tagged GltA::HA combined with either an empty vector control (pCtrl) or SPA-tagged vcdP over-expression plasmid, grown in LB medium to exponential phase (OD600 of 0.5). The SPA epitope contains the 3×FLAG and the calmodulin binding peptide sequences separated by a TEV protease cleavage site (Zeghouf et al, 2004). Therefore, protein samples corresponding to the total input and cell lysates before and after subjecting to immunoprecipitation with anti-Flag antibody were loaded on a SDS-PAGE gel. Western blotting with anti-HA and anti-Flag antibodies confirmed the interaction of GltA with VcdP in vivo. RNAP served as loading control. The solid triangles indicate the corresponding protein sizes.


B . Citrate synthase enzyme activity measurements performed on cellular lysates . An empty vector control ( pCtrl ) or vcdP expression plasmids ( pVcdP and pVcdP * ) were conjugated into V . cholerae wild - type , ΔvcdRP , ΔgltA or F383A GltA backgrounds . Cells were grown in LB medium to stationary phase and subsequently lysed . The protein concentrations of these cellular extracts were measured , and served as input for the assay . The resulting colorimetric product at 412nm was proportional to the enzymatic activity of citrate synthase present . The enzyme kinetics were assayed at 15 second intervals for at least 30 minutes and the activity per milligram of lysate was determined from the initial velocities .

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B. Citrate synthase enzyme activity measurements performed on cellular lysates. An empty vector control (pCtrl) or vcdP expression plasmids (pVcdP and pVcdP*) were conjugated into V. cholerae wild-type, ΔvcdRP, ΔgltA or F383A GltA backgrounds. Cells were grown in LB medium to stationary phase and subsequently lysed. The protein concentrations of these cellular extracts were measured, and served as input for the assay. The resulting colorimetric product at 412nm was proportional to the enzymatic activity of citrate synthase present. The enzyme kinetics were assayed at 15 second intervals for at least 30 minutes and the activity per milligram of lysate was determined from the initial velocities.


A , B After grown in nutrient rich medium to log phase , wild - type ( WT ) cells were treated with rapamycin ( 10 μM ) or subject to nitrogen starvation ( SD - N ) for 3 h and 16 h and degradation of autophagic marker GFP - Atg8 was detected . The cycloheximide ( CHX , 100 μg / mL , ) or thiolutin ( 10 μg / mL ) was simultaneously used to block protein synthesis or gene transcription .

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A, B After grown in nutrient rich medium to log phase, wild-type (WT) cells were treated with rapamycin (10 μM) or subject to nitrogen starvation (SD-N) for 3 h and 16 h and degradation of autophagic marker GFP-Atg8 was detected. The cycloheximide (CHX, 100 μg/mL,) or thiolutin (10 μg/mL) was simultaneously used to block protein synthesis or gene transcription.


Yeast cells were treated with thiolutin ( 10 μg / mL ) together with rapamycin or nitrogen starvation in different order for 3 h , 6h and 16 h and degradation of autophagic marker GFP - Atg8 was detected .

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Yeast cells were treated with thiolutin (10 μg/mL) together with rapamycin or nitrogen starvation in different order for 3 h, 6h and 16 h and degradation of autophagic marker GFP-Atg8 was detected.


B N - terminally GFP - tagged 50Q was checked for its autophagic degradation in indicated yeast cells by GFP processing assays after 1 , 4 and 16 h starvation . Yeast cells with ATG1 deletion were used as positive controls .

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B N-terminally GFP-tagged 50Q was checked for its autophagic degradation in indicated yeast cells by GFP processing assays after 1, 4 and 16 h starvation. Yeast cells with ATG1 deletion were used as positive controls.


H Vacuole localization and degradation of endocytosis substrate GFP - Sna3 in WT , atg1 ∆ and rpb9 ∆ cells . Scale bars : 5 μm .

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H Vacuole localization and degradation of endocytosis substrate GFP-Sna3 in WT, atg1∆ and rpb9∆ cells. Scale bars: 5 μm.


I Carboxypeptidases Y ( Cpy1 ) , a vacuolar - resident hydrolases , was analyzed for its transporting to vacuoles by the secretory pathway in WT , atg1 ∆ and rpb9 ∆ cells .

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I Carboxypeptidases Y (Cpy1), a vacuolar-resident hydrolases, was analyzed for its transporting to vacuoles by the secretory pathway in WT, atg1∆ and rpb9∆ cells.


E - I Overexpression of ATG1 could partially restore autophagy in rpb9 ∆ cells . ATG1 , ATG9 , ATG13 , ATG17 and ATG5 were overexpressed in rpb9 ∆ yeast cells , GFP - 50Q was checked for its autophagic degradation by GFP processing assays after 1 , 4 and 16 h starvation .

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E-I Overexpression of ATG1 could partially restore autophagy in rpb9∆ cells. ATG1, ATG9, ATG13, ATG17 and ATG5 were overexpressed in rpb9∆ yeast cells, GFP-50Q was checked for its autophagic degradation by GFP processing assays after 1, 4 and 16 h starvation.


B Indicated Rpb9 truncates and mutants were expressed in rpb9 ∆ cells and autophagic degradation of GFP - 50Q was checked by GFP - processing assays .

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B Indicated Rpb9 truncates and mutants were expressed in rpb9∆ cells and autophagic degradation of GFP-50Q was checked by GFP-processing assays.


E Purified GST - Gcn4 and His - Rpb9 proteins from E . coli cells were mixed and GST pulldown assays were performed .

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E Purified GST-Gcn4 and His-Rpb9 proteins from E. coli cells were mixed and GST pulldown assays were performed.


F - G Neither overexpression of Gcn4 in rpb9 ∆ yeast cells nor overexpression of Rpb9 in gcn4 ∆ yeast cells can restore autophagy . Gcn4 or Rpb9 were overexpressed in rpb9 ∆ or gcn4 ∆ yeast cells respectively and autophagic degradation of GFP - 50Q was detected .

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F-G Neither overexpression of Gcn4 in rpb9∆ yeast cells nor overexpression of Rpb9 in gcn4∆ yeast cells can restore autophagy. Gcn4 or Rpb9 were overexpressed in rpb9∆ or gcn4∆ yeast cells respectively and autophagic degradation of GFP-50Q was detected.


C Rpb9 orthologs from complex eukaryotic species could restore autophagy in rpb9 ∆ yeast cells . Indicated Rpb9 orthologs were expressed in rpb9 ∆ yeast cells and autophagic degradation of GFP - 50Q was detected .

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C Rpb9 orthologs from complex eukaryotic species could restore autophagy in rpb9∆ yeast cells. Indicated Rpb9 orthologs were expressed in rpb9∆ yeast cells and autophagic degradation of GFP-50Q was detected.


D N - terminal Zinc finger domain and linker region of Rpb9 orthologs from different species could restore autophagy defects in rpb9 ∆ yeast cells . Indicated truncates of Rpb9 orthologs were expressed in rpb9 ∆ yeast cells and autophagic degradation of GFP - 50Q was detected .

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D N-terminal Zinc finger domain and linker region of Rpb9 orthologs from different species could restore autophagy defects in rpb9∆ yeast cells. Indicated truncates of Rpb9 orthologs were expressed in rpb9∆ yeast cells and autophagic degradation of GFP-50Q was detected.


Growth inhibitory concentration ( GI50 ) of CY and bendamustine in the NCI60 cell line panel .

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Growth inhibitory concentration (GI50) of CY and bendamustine in the NCI60 cell line panel.


Nontoxic dose of Cpd A enhances bendamustine cytotoxicity in cancer cells . Images were taken at 48 h posttreatment .

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Nontoxic dose of Cpd A enhances bendamustine cytotoxicity in cancer cells. Images were taken at 48 h posttreatment.


Cpd A augments DNA damage caused by bendamustine . H1650 cells were treated with 100 μM Bend , 50 μM Cpd A , 100 μM Bend plus 50 μM Cpd A , or 10 μM CY for 12 h , and cell lysates were blotted for γ - H2AX . Actin was used as a loading control .

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Cpd A augments DNA damage caused by bendamustine. H1650 cells were treated with 100 μM Bend, 50 μM Cpd A, 100 μM Bend plus 50 μM Cpd A, or 10 μM CY for 12 h, and cell lysates were blotted for γ-H2AX. Actin was used as a loading control.


Upper panel , in vitro HDAC1 inhibition assay using bendamustine and CY . Lower panel , summary of CY IC50 against other HDACs .

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Upper panel, in vitro HDAC1 inhibition assay using bendamustine and CY. Lower panel, summary of CY IC50 against other HDACs.


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CY and CpdA , but not bendamustine or CpdB , inhibited HDAC activities in cancer cells . H1650 cells were treated with 20 μM CY , 20 μM CpdA , 200 μM CpdB , 200 μM Bend , or 10 μM SAHA for 12 h , and cell lysates were blotted for histone acetylation markers . Actin was used as a loading control .

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CY and CpdA, but not bendamustine or CpdB, inhibited HDAC activities in cancer cells. H1650 cells were treated with 20 μM CY, 20 μM CpdA, 200 μM CpdB, 200 μM Bend, or 10 μM SAHA for 12 h, and cell lysates were blotted for histone acetylation markers. Actin was used as a loading control.