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e, f, GLI1 (*P = 0.04, n = 3) (e) and GLI2 (***P = 0.0005, **P = 0.0046, †P = 0.0132, n = 3) (f) messenger RNA expression in Atg5−/− MEFs.


g , h , IFT20 immunoblot in Atg5 − / − ( g ) WT MEFs with lysosomal inhibitors ( NL ) ( h ) .

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g, h, IFT20 immunoblot in Atg5−/− (g) and WT MEFs with lysosomal inhibitors (NL) (h).


i , IFT20 immunoblot in cytosol ( Cyt ) , homogenate ( Hom ) autophagosomes ( APG ) . j , IFT20 in Atg5 − / − MEFs ( * P = 0 . 038 , n = 6 ) . k , IFT20 immunoblot in Atg5 − / − MEFs . n . s . , statistically non - significant . Mean ± s . d . in d j mean ± s . e . m . in other panels .

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i, IFT20 immunoblot in cytosol (Cyt), homogenate (Hom) and autophagosomes (APG). j, IFT20 protein levels in Atg5−/− MEFs (*P = 0.038, n = 6). k, IFT20 immunoblot in Atg5−/− MEFs. n.s., statistically non-significant. Mean ± s.d. in d and j and mean ± s.e.m. in other panels.


( A ) Breast cancer lines were sorted using CD44 for luminal MCF - 7 ( left panel ) or EpCAM for MDA - MB - 231 ( right panel ) . Serial mammosphere assays were performed using 4000 for each fraction , counting spheres after 3 . Plots show mean ± of mammospheres formed with 4000 from 3 independent experiments performed in triplicate .

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(A) Breast cancer cell lines were sorted using CD44 for luminal MCF-7 cells (left panel) or EpCAM for MDA-MB-231 cells (right panel). Serial mammosphere assays were performed using 4000 cells for each cell fraction, counting spheres after 3 weeks. Plots show the mean ± SEM of mammospheres formed with 4000 cells from 3 independent experiments performed in triplicate.


( B ) Extreme limiting dilution ( ELDA ) of MCF - 7 . Sorted were serially diluted ( 1250 , 625 , 312 , 156 78 ) then seeded in 96 - well plates . After one week , were fixed , stained counted . lower limit for well repopulation in CD44 + was 312 , compared with 615 for both unsorted CD44 - populations . data obtained were evaluated using ELDA software . p values that single - hit hypothesis using likelihood are shown . curves are depicted in Fig

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(B) Extreme limiting dilution assay (ELDA) of MCF-7 cells. Sorted cells were serially diluted (1250, 625, 312, 156 and 78 cells) and then seeded in 96-well plates. After one week, the cells were fixed, stained and counted. The lower limit for well repopulation in CD44+ cells was 312 cells, compared with 615 cells for both unsorted and CD44- populations. The data obtained were evaluated using the ELDA software. p values that test the single-hit hypothesis using the likelihood ratio test are shown. The curves are depicted in Fig EV1


( C ) Relative expression of stemness factors SOX2 OCT4 EMT markers SNAIL , Vimentin TWIST in MCF - 7 / CD44 + . fold change was calculated using GAPDH as an internal control using 2 - ΔΔCT method comparing or non - versus total . box whiskers graph shows range , median quartile interval values from 3 independent experiments .

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(C) Relative expression of the stemness factors SOX2 and OCT4 and the EMT markers SNAIL, Vimentin and TWIST in MCF-7/CD44+ cells. The fold change was calculated using GAPDH as an internal control using the 2-ΔΔCT method comparing stem or non-stem cells versus the total cell line. The box and whiskers graph shows the range, median and quartile interval values from 3 independent experiments.


( D ) miR - 10b overexpression affecting stemness EMT factors . Relative of miR - 10b ( relative to RNU6B ) , HOXD10 RHOC ( relative to GAPDH ) was performed in MCF - 7 / CD44 + fractions compared to non - enriched . Box plots show range , median quartile interval values from 3 independent experiments . A two - Student ' s t was used to compare two groups ( p < 0 . 05 ) . , standard error of mean ; TLDA , TaqMan Low Density Arrays ; EMT , epithelial - mesenchymal transition .

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(D) miR-10b overexpression affecting stemness and EMT factors. Relative quantification of miR-10b (relative to RNU6B), HOXD10 and RHOC (relative to GAPDH) was performed in MCF-7/CD44+ fractions compared to non-enriched cells. Box plots show the range, median and quartile interval values from 3 independent experiments. A two-tailed Student's t test was used to compare the two groups (p < 0.05). SEM, standard error of the mean; TLDA, TaqMan Low Density Arrays; and EMT, epithelial-mesenchymal transition.


( E ) Relative expression of stemness factors SOX2 OCT4 EMT markers SNAIL Vimentin in MDA - MB - 231 / EpCAM + . fold change was calculated using GAPDH as an internal control using 2 - ΔΔCT method comparing or non - versus total . box whiskers graph shows range , median quartile interval values from 3 independent experiments .

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(E) Relative expression of stemness factors SOX2 and OCT4 and EMT markers SNAIL and Vimentin in MDA-MB-231/EpCAM+ cells. The fold change was calculated using GAPDH as an internal control using the 2-ΔΔCT method comparing stem or non-stem cells versus the total cells. The box and whiskers graph shows the range, median and quartile interval values from 3 independent experiments.


( F ) Extreme Limiting Dilution ( ELDA ) of MDA - MB - 231 . Sorted were serially diluted ( 1250 , 625 , 312 , 156 78 ) then seeded in 96 - well plates . After one week , were fixed , stained counted . lower limit for well repopulation in EpCAM + was 156 compared with 625 for both unsorted EpCAM - populations . data obtained were evaluated using ELDA software . p values that single - hit hypothesis using likelihood are shown . curves are depicted in Fig

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

(F) Extreme Limiting Dilution Assay (ELDA) of MDA-MB-231 cells. Sorted cells were serially diluted (1250, 625, 312, 156 and 78 cells) and then seeded in 96-well plates. After one week, the cells were fixed, stained and counted. The lower limit for well repopulation in EpCAM+ cells was 156 cells compared with 625 cells for both unsorted and EpCAM- populations. The data obtained were evaluated using the ELDA software. p values that test the single-hit hypothesis using the likelihood ratio test are shown. The curves are depicted in Fig EV1


( G ) Nine miRNAs are commonly regulated in both CD44 + EpCam + breast cancer . Quantitative RT - PCR of miRNAs was performed on sorted MCF - 7 MDA - MB - 231 populations using TaqMan Low Density Arrays ( TLDA ) . Results were plotted as a heat map comparing CD44 + to CD44 - ( MCF - 7 ) EpCam + to EpCam - . scale is shown below map .

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(G) Nine miRNAs are commonly regulated in both CD44+ and EpCam+ breast cancer cells. Quantitative RT-PCR of miRNAs was performed on sorted MCF-7 and MDA-MB-231 cell populations using TaqMan Low Density Arrays (TLDA). Results were plotted as a heat map comparing CD44+ to CD44- cells (MCF-7) and EpCam+ to EpCam- cells. The scale is shown below the map.


( A ) Upper panel , left : MCF - 7 / CD44 + were transfected with anti - miR - 10b or a scrambled control , qRT - PCR was performed . Upper panel , right : An aliquot of these was subjected to mammosphere - forming assays . graph shows mean ± of number of formed mammospheres per 4000 seeded in 3 independent experiments . Lower panel , left : Expression of miR - 10b relative to RNU6B in MCF - 7 stably transfected with a plasmid containing miR - 10b gene versus an empty vector . Results are mean ± values from 3 independent experiments . Lower panel , right : miR - 10b - overexpressing MCF - 7 analyzed by serial mammosphere assays . mean ± of number of formed mammospheres per 4000 seeded in 3 independent experiments was plotted on each bar .

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(A) Upper panel, left: MCF-7/CD44+ cells were transfected with anti-miR-10b or a scrambled control, and qRT-PCR was performed. Upper panel, right: An aliquot of these cells was subjected to mammosphere-forming assays. The graph shows the mean ± SEM of the number of formed mammospheres per 4000 seeded cells in 3 independent experiments. Lower panel, left: Expression of miR-10b relative to RNU6B in MCF-7 cells stably transfected with a plasmid containing the miR-10b gene versus an empty vector. Results are the mean ± SEM values from 3 independent experiments. Lower panel, right: miR-10b-overexpressing MCF-7 cells analyzed by serial mammosphere assays. The mean ± SEM of the number of formed mammospheres per 4000 seeded cells in 3 independent experiments was plotted on each bar.


( B ) Upper panel : Mammosphere formation was performed by seeding 20 , 000 MDA - MB - 231 / EpCAM + , after 3 , were disaggregated , counted , reseeded . Stable miR - 10b - overexpressing from 3 independent clones were tested in 3 passages , number was plotted as mean ± .

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(B) Upper panel: Mammosphere formation was performed by seeding 20,000 MDA-MB-231/EpCAM+ cells, and after 3 weeks, the cells were disaggregated, counted, and reseeded. Stable miR-10b-overexpressing cells from 3 independent clones were tested in 3 passages, and cell number was plotted as the mean ± SEM.


( C ) In vivo limiting dilution assays . miR - 10 - OE or control were subcutaneously injected in backs of mice at 3 doses ( 1 , 000 , 10 , 000 50 , 000 for MCF - 7 5000 , 10 000 20 000 for SKBR - 3 ) . Mice were sacrificed after eight , burden was analyzed using ELDA software . Plots are depicted in Fig EV2 . tables show number of mice with tumors versus total number of mice injected . Below tables , estimated frequency ( freq ) of for each group p is shown . frequency for MCF - 7 was 1 in 34252 ( range 94888 - 12364 ) in miR - 10b - OE versus 1 in 238965 ( Range 1817801 - 31414 ) in miR - scr . frequency for SKBR3 was 1 in 5013 ( Range 11135 - 2257 ) in miR - 10b - OE versus 1 in 18778 ( Range 45397 - 7767 ) in miR - scr .

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(C) In vivo limiting dilution assays. miR-10-OE or control cells were subcutaneously injected in the backs of mice at 3 doses (1,000, 10,000 and 50,000 cells for MCF-7 and 5000, 10 000 and 20 000 for SKBR-3). Mice were sacrificed after eight weeks, and tumor burden was analyzed using the ELDA software. Plots are depicted in Fig EV2. The tables show the number of mice with tumors versus the total number of mice injected. Below the tables, the estimated frequency (freq) of stem cells for each group and p is shown. Stem cell frequency for MCF-7 cells was 1 in 34252 (range 94888-12364) in miR-10b-OE cells versus 1 in 238965 (Range 1817801-31414) in miR-scr cells. Stem cell frequency for SKBR3 cells was 1 in 5013 (Range 11135-2257) in miR-10b-OE cells versus 1 in 18778 (Range 45397-7767) in miR-scr cells.


( D ) Stemness EMT marker mRNA following miR - 10b modulation . miR - 10b , OCT4 / 3 , SNAIL Vimentin expression was quantified in breast cancer lines using transient transfection of miR - 10b versus a miR - Scr vector ( left ) or anti - miR - 10b versus anti - miR - ctl ( right ) . Log - scale plots show mean for relative expression of each gene from triplicate independent transient transfections , each performed three times . A Mann - Whitney U was used to compare expression between groups . SOX2 , OCT4 Vimentin were significantly modulated by up - or down - regulation of miR - 10b ( p = 0 . 0043 , p = 0 . 0054 p = 0 . 000908 , respectively ) . , standard error of mean .

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(D) Stemness and EMT marker mRNA levels following miR-10b modulation. miR-10b, OCT4/3, SNAIL and Vimentin expression was quantified in breast cancer cell lines using transient transfection of miR-10b versus a miR-Scr vector (left) or anti-miR-10b versus anti-miR-ctl (right). Log-scale plots show the mean and SEM for the relative expression of each gene from triplicate independent transient transfections, each performed three times. A Mann-Whitney U test was used to compare expression between groups. SOX2, OCT4 and Vimentin were significantly modulated by up- or down-regulation of miR-10b (p=0.0043, p=0.0054 and p=0.000908, respectively). SEM, standard error of the mean.


( B ) MCF - 7 , SKBR - 3 T47 - D breast cancer were transfected with gain - or loss - of - function constructs / shRNAs their respective controls ( a miR - 10b - OE plasmid or anti - miR - 10b ) . MREs of validated target HOXD10 of putative targets PTEN PIK3CA were cloned into pMIR - Report , dual luciferase reporter assays were performed . A mutated control miR - 10b ( MRE MUT PTEN ) was also included . Bars show mean ± from 6 independent transfections .

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(B) MCF-7, SKBR-3 and T47-D breast cancer cells were transfected with gain- or loss-of-function constructs/shRNAs and their respective controls (a miR-10b-OE plasmid or anti-miR-10b). MREs of the validated target HOXD10 and of the putative targets PTEN and PIK3CA were cloned into pMIR-Report, and dual luciferase reporter assays were performed. A mutated control miR-10b (MRE MUT PTEN) was also included. Bars show the mean ± SEM from 6 independent transfections.


( C ) PTEN PIK3CA expression after miR - 10b modulation . Left panel shows miR - 10b , PTEN PIK3CA expression by qRT - PCR in MCF - 7 with ectopic miR - 10b expression . Right panel shows miR - 10b , PTEN PIK3CA expression by qRT - PCR in MDA - MB - 231 after antagomiR - 10b transfection . Data are from three independent transient transfections performed by triplicate .

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(C) PTEN and PIK3CA expression after miR-10b modulation. Left panel shows miR-10b, PTEN and PIK3CA expression by qRT-PCR in MCF-7 cells with ectopic miR-10b expression. Right panel shows miR-10b, PTEN and PIK3CA expression by qRT-PCR in MDA-MB-231 cells after antagomiR-10b transfection. Data are from three independent transient transfections performed by triplicate.


( D ) To further confirm PTEN targeting , we performed gain - or loss - of - function experiments in breast cancer lines ( MCF - 7 , MCF - 7 / CD44 + , SKBR - 3 T47D ) by overexpressing miR - 10b or inhibiting it with a miR - 10b - OE plasmid or anti - miR - 10b shRNAs . PTEN miR - 10b were measured by qRT - PCR . log2 - fold change of PTEN ( left panel ) PIK3CA ( right panel ) mRNA obtained through 2 - ΔΔCT method were plotted against their respective miR - 10b expression . Correlation was performed [ PTEN ( r = - 0 . 80 ) , PI3KCA ( r = 0 . 43 ) ] . A two - Student ' s t was used to compare two groups ( * p < 0 . 05 ) . , standard error of mean .

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(D) To further confirm PTEN targeting, we performed gain- or loss-of-function experiments in breast cancer cell lines (MCF-7, MCF-7/CD44+, SKBR-3 and T47D) by overexpressing miR-10b or inhibiting it with a miR-10b-OE plasmid or anti-miR-10b shRNAs. PTEN and miR-10b levels were measured by qRT-PCR. The log2-fold change of PTEN (left panel) and PIK3CA (right panel) mRNA levels obtained through the 2-ΔΔCT method were plotted against their respective miR-10b expression levels. Correlation analysis was performed [PTEN (r=-0.80), PI3KCA (r=0.43)]. A two-tailed Student's t test was used to compare the two groups (*p < 0.05). SEM, standard error of the mean.


( E ) PTEN modulation by miR - 10b . Upper panel : PTEN in SKBR - 3 , MCF - 7 were analyzed by Western blot . GAPDH was used as a loading control with cultured as in ( B ) . Lower panel : Densitometric of Western blot assays performed with three biological replicates .

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(E) PTEN protein modulation by miR-10b. Upper panel: PTEN protein levels in SKBR-3, and MCF-7 cells were analyzed by Western blot. GAPDH was used as a loading control with cells cultured as in (B). Lower panel: Densitometric analysis of Western blot assays performed with three biological replicates.


( F ) PTEN immunohistochemistry of MCF - 7 - miR - 10b - OE or control MCF - 7 - miR - Scr - OE tumorxenografts .

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(F) PTEN immunohistochemistry analysis of MCF-7-miR-10b-OE or control MCF-7-miR-Scr-OE tumorxenografts.


( G ) miR - 10b overexpression does not increase mammosphere formation in PTEN - depleted . MCF - 7 - miR - Scr - OE ( left panels ) MCF - 7 - miR - 10b - OE ( right panels ) were transfected with a Dicer - substrate directed toward PTEN , after 24 h , quantitative RT - PCR assays for PTEN , SOX2 , OCT4 , SNAIL Vimentin were performed . Additionally , same were analyzed for mammosphere formation . graph shows mean standard error of relative increase of each marker or number of mammospheres in PTEN - depleted ( black bars ) in transfected with a scrambled control ( bars ) in three independent biological experiments performed by triplicate .

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(G) miR-10b overexpression does not increase mammosphere formation in PTEN-depleted cells. MCF-7-miR-Scr-OE cells (left panels) and MCF-7-miR-10b-OE (right panels) were transfected with a Dicer-substrate RNA directed toward PTEN, and after 24 h, quantitative RT-PCR assays for PTEN, SOX2, OCT4, SNAIL and Vimentin were performed. Additionally, the same cells were analyzed for mammosphere formation. The graph shows the mean and standard error of the relative increase of each marker or number of mammospheres in PTEN-depleted cells (black bars) and in cells transfected with a scrambled control (white bars) in three independent biological experiments performed by triplicate.


( H ) T47 - D were transfected with antagomiR - 10b , a dsi - for PTEN or both mammospheres produced as described in material methods . Bars show mean from three independent experiments . * p = 0 . 003 by Student ´ s T , after ANOVA showing statistical differences in groups .

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(H) T47-D cells were transfected with antagomiR-10b, a dsi-RNA for PTEN or both and mammospheres produced as described in material and methods. Bars show mean and SEM from three independent experiments. * p=0.003 by Student´s T test, after ANOVA showing statistical differences in groups.


( I ) PTEN overexpression compensates for miR - 10b overexpression . Left panel : MDA - 468 were transfected with a plasmid overexpressing wild - type PTEN or a version containing a mutated MRE in its 3 ' - UTR , with or without a plasmid containing miR10b PTEN mRNA expression was measured by end - point RT - PCR . Middle panel : Relative expression of stemness factor SNAIL . plots show relative expression from biological triplicate analyses ( mean SE shown ) . Right panel : Relative expression of stemness factor SOX2 . plots show relative expression from biological triplicate analyses ( mean SE shown ) .

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(I) PTEN overexpression compensates for miR-10b overexpression. Left panel: MDA-468 cells were transfected with a plasmid overexpressing wild-type PTEN or a version containing a mutated MRE in its 3'-UTR, with or without a plasmid containing miR10b and PTEN mRNA expression was measured by end-point RT-PCR. Middle panel: Relative expression of stemness factor SNAIL. The plots show the relative expression from biological triplicate analyses (mean and SE shown). Right panel: Relative expression of stemness factor SOX2. The plots show the relative expression from biological triplicate analyses (mean and SE shown).


( A ) AKT phosphorylation in CSCs from MCF - 7 sorted by CD44 . GAPDH were used as a loading control .

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(A) AKT phosphorylation in CSCs from MCF-7 cells sorted by CD44. GAPDH protein levels were used as a loading control.


( B ) Upper panel . Cultures were serum - starved for 18 h then stimulated with insulin or exposed to PI3K pathway inhibitors Wortmannin or LY294002 . PTEN , PI3KCA , GSK3 phospho - AKT Ser - 473 were analyzed by Western blot . GAPDH was used as a loading control . AKT kinase activity was analyzed in these by immunoprecipitating phosphorylated AKT with a p - AKT - Ser - 473 antibody using chimeric GSK - 3 as a substrate . A two - Student ' s t was used to compare two groups ( p < 0 . 05 ) . , standard error of mean . Lower panel . SKBR3 were transiently transfected with a plasmid containing an open reading frame of PTEN , miR - 10b or both phospho - AKT , AKT or tubulin analyzed by Western Blot assays . Control is empty vector - transfected .

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

(B) Upper panel. Cultures were serum-starved for 18 h and then stimulated with insulin or exposed to the PI3K pathway inhibitors Wortmannin or LY294002. PTEN, PI3KCA, GSK3 and phospho-AKT Ser-473 protein levels were analyzed by Western blot. GAPDH was used as a loading control. AKT kinase activity was analyzed in these cells by immunoprecipitating phosphorylated AKT proteins with a p-AKT-Ser-473 antibody and using chimeric GSK-3 as a substrate. A two-tailed Student's t test was used to compare the two groups (p<0.05). SEM, standard error of the mean. Lower panel. SKBR3 cells were transiently transfected with a plasmid containing an open reading frame of PTEN, miR-10b or both and phospho-AKT, AKT or tubulin levels analyzed by Western Blot assays. Control is empty vector-transfected cells.


( C ) Clonogenic ability of miR - 10b - depleted CD44 + MCF - 7 after PI3K pathway inhibition . Cultures were serum - starved for 18 h then stimulated with insulin or exposed to PI3K pathway inhibitors wortmannin or LY294002 . transfected with anti - miR - 10b or a scrambled control ( anti - miR - ctl ) were seeded in soft agar at clonal density exposed to insulin or PIK3CA inhibitors . After 3 , colonies were quantified . Bars show mean ± for 3 independent experiments .

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(C) Clonogenic ability of miR-10b-depleted CD44+ MCF-7 cells after PI3K pathway inhibition. Cultures were serum-starved for 18 h and then stimulated with insulin or exposed to the PI3K pathway inhibitors wortmannin or LY294002. Cells transfected with anti-miR-10b or a scrambled control (anti-miR-ctl) were seeded in soft agar at clonal density and exposed to insulin or PIK3CA inhibitors. After 3 weeks, colonies were quantified. Bars show the mean ± SEM for 3 independent experiments.


( D ) miR - 10b overexpression does not induce additional changes in colony numbers in response to insulin or PI3K inactivation . MCF - 7 stable miR - Scr - OE or miR - 10b - OE were seeded in soft agar at clonal density exposed to insulin or PIK3CA inhibitors . After 3 , colonies were quantified . Bars show mean ± for 3 independent experiments .

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(D) miR-10b overexpression does not induce additional changes in cell colony numbers in response to insulin or PI3K inactivation. MCF-7 stable miR-Scr-OE or miR-10b-OE cells were seeded in soft agar at clonal density and exposed to insulin or PIK3CA inhibitors. After 3 weeks, colonies were quantified. Bars show the mean ± SEM for 3 independent experiments.


Module - trait relationships were established by WGCNA using eight independent microarray analyses comprising 375 human prostate samples . Gene modules ( y axis ) are denoted by an arbitrary color name . Bins show Pearson correlation value between gene expression of each module within noted phenotype / disease stage ( x axis ) p values . A value of 1 ( red ) quantifies strongest positive correlation ( genes are upregulated ) , - 1 ( blue ) strongest negative correlation ( genes are downregulated ) , 0 ( ) no correlation .

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Module-trait relationships were established by WGCNA using eight independent microarray analyses comprising 375 human prostate samples. Gene modules (y axis) are denoted by an arbitrary color name. Bins show the Pearson correlation value between gene expression levels of each module within the noted phenotype/disease stage (x axis) and p values. A value of 1 (red) quantifies the strongest positive correlation (genes are upregulated), -1 (blue) the strongest negative correlation (genes are downregulated), and 0 (white) no correlation.


Microarray was performed following doxycycline - regulated specific AR - V7 depletion ( using a tet - plKO backbone ) in 22Rv1 PC compared to doxycycline - treated shGFP controls . genes that were significantly regulated by shAR - V7 ( in either direction , p value < 0 . 05 ) were distributed among gene modules defined by WGCNA in panel A . Upregulated genes ( red ) are those in which expression decreased following AR - V7 depletion conversely downregulated genes ( blue ) are those that increased following AR - V7 depletion .

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Microarray analysis was performed following doxycycline-regulated specific AR-V7 depletion (using a tet-plKO backbone) in 22Rv1 PC cells compared to doxycycline-treated shGFP controls. The genes that were significantly regulated by shAR-V7 (in either direction, p value < 0.05) were distributed among the gene modules defined by WGCNA in panel A. Upregulated genes (red) are those in which expression decreased following AR-V7 depletion and conversely downregulated genes (blue) are those that increased following AR-V7 depletion.


Hornberg et al . , 2011 gene expression profiling array data was analyzed to determine expression of seven genes in human CRPC bone metastases , grouped by their relative of AR - VS , mainly AR - V7 . ( High - of AR - V7 ( top quartile ) or lower of AR - V7 ( quartiles 1 - 3 ) . Data are plotted as mean ± s . e . m . . Non - parametric Mann - Whitney was performed ( two - ) . Note that BUB1B expression was not measured in these microarrays . * * Significant at a p value < 0 . 05 . N ( AR - V7 low ) = 20 ; N ( AR - V7 high ) = 10 .

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Hornberg et al., 2011 gene expression profiling array data was analyzed to determine the expression levels of the seven genes in human CRPC bone metastases, grouped by their relative levels of AR-VS, mainly AR-V7. (High-levels of AR-V7 (top quartile) or lower levels of AR-V7 (quartiles 1-3). Data are plotted as the mean ± s.e.m.. Non-parametric Mann-Whitney test was performed (two-tailed). Note that BUB1B expression was not measured in these microarrays. ** Significant at a p value < 0.05. N (AR-V7 low) = 20; N (AR-V7 high) = 10.


Kaplan - Meier curves for Disease - Free Survival ( DFS ) overall survival were built using Prostate Adenocarcinoma TCGA dataset ( 465 samples ) ( upper graphs ) . black curves denote cases with normal expression of gene set , red represents cases where mRNA of seven genes were upregulated ( z - score threshold ≤ 1 . 96 ) . For DFS : p - value = 0 . 0009 ; for : p - value = 0 . 026 . An independent dataset was analyzed ( Prostate Adenocarcinoma MSKCC , Cancer 2010 , 123 samples ) ( lower graphs ) . black curves denote cases with normal expression of gene set , red represents cases where mRNA of at least five genes of gene set were upregulated ( z - score threshold ≤ 1 . 96 ) . For DFS : p - value = 0 . 0007 ; for : p - value = 0 . 00546 .

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The Kaplan-Meier curves for Disease-Free Survival (DFS) and overall survival were built using the Prostate Adenocarcinoma TCGA dataset (465 samples) (upper graphs). The black curves denote cases with normal expression of the gene set, and red represents cases where the mRNA levels of the seven genes were upregulated (z-score threshold ≤ 1.96). For DFS: p-value = 0.0009; for death: p-value = 0.026. An independent dataset was analyzed (Prostate Adenocarcinoma MSKCC, Cancer Cell 2010, 123 samples) (lower graphs). The black curves denote cases with normal expression of the gene set, and red represents cases where the mRNA levels of at least five genes of the gene set were upregulated (z-score threshold ≤ 1.96). For DFS: p-value = 0.0007; for death: p-value = 0.00546.


proliferation was examined in CRPC 22Rv1 following depletion of mRNAs for seven genes or shGFP controls , using shRNA against coding region for each gene ( shRNA # 2 ) . number was measured using a non - perturbing restricted dye quantified after 72 hours using Incucyte Zoom System . Data shown are mean ± s . e . m . of 8 to 12 replicates normalized to their shGFP control . Kruskal - Wallis ( p value < 0 . 0001 , two - ) Dunn ' s multiple comparisons were performed .

Cell proliferation was examined in the CRPC cell line 22Rv1 following individual depletion of mRNAs for the seven genes or shGFP controls, using shRNA against the coding region for each gene (shRNA #2). Cell number was measured using a non-perturbing nuclear restricted dye and quantified after 72 hours using Incucyte Zoom System. Data shown are mean ± s.e.m. of 8 to 12 replicates normalized to their shGFP control. Kruskal-Wallis test (p value < 0.0001, two-tailed) and Dunn's multiple comparisons test were performed.


Representative of 22Rv1 stably depleted of BUB1B , or control ( shGFP ) are shown .

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Representative images of 22Rv1 stably depleted of BUB1B, or control (shGFP) are shown.


22Rv1 stably depleted of each of seven genes were transfected with a dual plasmid luciferase reporter system which quantifies AR activity basal transcription . was conducted in 2 % CSS to measure AR ligand - independent transcriptional activity . Data represent two independent experiments performed in triplicate , showing mean ± s . e . m . , normalized to their shGFP controls . Kruskal - Wallis ( p value < 0 . 0001 , two - ) Dunn ' s multiple comparisons were performed . * Significant at a p value < 0 . 05 , * * p value < 0 . 001 .

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22Rv1 stably depleted of each of the seven genes were transfected with a dual plasmid luciferase reporter system which quantifies AR activity and basal transcription. The assay was conducted in 2% CSS to measure AR ligand-independent transcriptional activity. Data represent two independent experiments performed in triplicate, showing the mean ± s.e.m., and normalized to their shGFP controls. Kruskal-Wallis test (p value < 0.0001, two-tailed) and Dunn's multiple comparisons test were performed. * Significant at a p value < 0.05, ** p value < 0.001.


expression of FKBP5 UBE2C determined by RT - qPCR normalized to GAPDH mRNA was examined in 22Rv1 stably expressing shRNA for each of seven genes . were cultured in 2 % CSS . Data represent two independent experiments performed in duplicate or triplicate , showing mean ± s . e . m . , normalized to their shGFP controls . Kruskal - Wallis ( p value < 0 . 0001 , two - ) Dunn ' s multiple comparisons were performed . * Significant at a p value < 0 . 05 , * * p value < 0 . 001 .

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

The expression of FKBP5 and UBE2C determined by RT-qPCR analysis and normalized to GAPDH mRNA levels was examined in 22Rv1 cells stably expressing shRNA for each of the seven genes. Cells were cultured in 2% CSS. Data represent two independent experiments performed in duplicate or triplicate, showing the mean ± s.e.m., and normalized to their shGFP controls. Kruskal-Wallis test (p value < 0.0001, two-tailed) and Dunn's multiple comparisons test were performed. * Significant at a p value < 0.05, ** p value < 0.001.


CRPC 22Rv1 was cultured in 2 % CSS media was treated for 72 hours with vehicle ( DMSO ) , doxorubicin ( DOX ) , N9 - isopropylolomoucine ( N - 9 ) , or combination of DOX N - 9 at different concentrations . confluence was monitored using Incucyte Zoom System experiments were done with eight replicates each . data were analyzed using Compusyn software , a normalized isobologram was built . table shows Combination Index ( CI ) for different drug combinations . CI = 1 represents additivity , CI < 1 synergism , CI > 1 antagonistic effects .

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The CRPC cell line 22Rv1 was cultured in 2% CSS media and was treated for 72 hours with vehicle (DMSO), doxorubicin (DOX), N9-isopropylolomoucine (N-9), or the combination of DOX and N-9 at different concentrations. Cell confluence was monitored using Incucyte Zoom System and the experiments were done with eight replicates each. The data were analyzed using Compusyn software, and a normalized isobologram was built. The table shows the Combination Index (CI) for the different drug combinations. CI=1 represents additivity, CI<1 synergism, and CI>1 antagonistic effects.


non - tumorigenic prostate epithelial RWPE1 , AR - null PC PC3 , androgen dependent LNCaP , CRPC lines C4 - 2B 22Rv1 were treated for 72 hours with vehicle ( DMSO ) , DOX ( 100 ng / mL [ 184 nM ] ) , N - 9 ( 200 ng / mL [ 613 nM ] ) , or combination of DOX ( 100 ng / mL [ 184 nM ] ) N - 9 ( 200 ng / mL [ 613 nM ] ) . C4 - 2B 22Rv1 were kept in 10 % CSS media , other lines in 10 % FBS . confluence was monitored using Incucyte Zoom System . Data represent two independent experiments , with four to six replicates each , showing mean ± s . e . m . , normalized to vehicle controls ( Kruskal - Wallis , P value < 0 . 0001 , two - ) . * Significant at a p value < 0 . 05 , * * p value < 0 . 01 , * * * p value < 0 . 001 .

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The non-tumorigenic prostate epithelial cell line RWPE1, the AR-null PC cell line PC3, the androgen dependent cell line LNCaP, and the CRPC cell lines C4-2B and 22Rv1 were treated for 72 hours with vehicle (DMSO), DOX (100 ng/mL [184 nM]), N-9 (200 ng/mL [613 nM]), or the combination of DOX (100 ng/mL [184 nM]) and N-9 (200 ng/mL [613 nM]). C4-2B and 22Rv1 cells were kept in 10% CSS media, and the other cell lines in 10% FBS. Cell confluence was monitored using the Incucyte Zoom System. Data represent two independent experiments, with four to six replicates each, showing the mean ± s.e.m., and normalized to vehicle controls (Kruskal-Wallis test, P value <0.0001, two-tailed). *Significant at a p value < 0.05, ** p value < 0.01, *** p value < 0.001.


non - tumorigenic prostate RWPE - 1 CRPC 22Rv1 were treated for 72 hours with vehicle ( DMSO ) or combination of DOX N - 9 at 100 ng / mL N - 9 200 ng / mL , respectively . confluence was monitored using Incucyte Zoom System .

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The non-tumorigenic prostate cell line RWPE-1 and the CRPC cell line 22Rv1 were treated for 72 hours with vehicle (DMSO) or the combination of DOX and N-9 at 100 ng/mL and N-9 200 ng/mL, respectively. Cell confluence was monitored using the Incucyte Zoom System.


( A ) Distribution of antimycin A resistance scores for wild isolates , compared to standard laboratory ( red vertical ) . Resistance scores are of fitness on rich glucose media with vs without 500 µg / L antimycin A . Fitness was estimated on colony on solid media , corrected for spatial plate effects After quality control , we obtained quantitative fitness scores for 154 strains , with a signal - to - noise of 29 . 8 an unexplained variance of 0 . 12 .

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(A) Distribution of antimycin A resistance scores for wild isolates, compared to standard laboratory strain (red vertical line). Resistance scores are the ratio of fitness on rich glucose media with vs without 500 µg/L antimycin A. Fitness was estimated based on colony size on solid media, corrected for spatial and plate effects After quality control, we obtained quantitative fitness scores for 154 strains, with a signal-to-noise ratio of 29.8 and an unexplained variance of 0.12.


( B ) Volcano plot of a genome - wide association using mixed - model linear regression of antimycin A resistance for 118 , 527 genetic variants . Variants with moderate or high impact are shown in blue . Red dot : variant at locus I : 3845516 , which causes a T343A change in Pyk1 - acid . This variant was among top scoring ( effect = - 0 . 645 , rank 34 ; p = 0 . 0008 , rank 70 ) .

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(B) Volcano plot of a genome-wide association using mixed-model linear regression of antimycin A resistance for 118,527 genetic variants. Variants with moderate or high impact are shown in blue. Red dot: the variant at locus I:3845516, which causes a T343A change in the Pyk1 amino-acid sequence. This variant was among the top scoring (effect size=-0.645, rank 34; p=0.0008, rank 70).


( C ) Boxplot showing antimycin A resistance for 154 strains grouped by two alleles at pyk1 locus . Strains carrying a C at this genomic locus ( orange box ) generally have higher antimycin resistance than strains carrying reference allele T ( blue box ) . As is standard , this boxplot other boxplots in this manuscript show median of data as central , quartiles as box extend of of distribution as whiskers . Points which are 1 . 5 times inter - quartile range beyond high low quartiles are considered outliers shown individually .

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(C) Boxplot showing antimycin A resistance for 154 strains grouped by the two alleles at the pyk1 locus. Strains carrying a C at this genomic locus (orange box) generally have higher antimycin resistance than strains carrying the reference allele T (blue box). As is standard, this boxplot and all other boxplots in this manuscript show the median of the data as central line, the quartiles as box and the extend of the rest of the distribution as whiskers. Points which are 1.5 times the inter-quartile range beyond the high and low quartiles are considered outliers and shown individually.


( B ) Left boxplot : two glycolytic intermediates directly upstream of PYK were strongly depleted in A - ( nT - = 9 , nA - = 8 ) . Right barplot : PYK activity was directly measured using a lactate dehydrogenase - coupled colorimetric enzyme activity , showing mean standard deviation of substrate conversion rate for 3 biological replicates , each measured in technical duplicates . Data information : Significance keys : * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 ( Welch ' s t - )

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(B) Left boxplot: two glycolytic intermediates directly upstream of PYK were strongly depleted in the A-strain (nT-strain=9, nA-strain=8). Right barplot: PYK activity was directly measured using a lactate dehydrogenase-coupled colorimetric enzyme activity assay, showing the mean and standard deviation of substrate conversion rate for 3 biological replicates, each measured in technical duplicates. Data information: Significance keys: * p<0.05, ** p<0.005, *** p<0.0005 (Welch's t-test)


( C ) Concentrations of fructose - 1 , 6 - bisphosphate , which correlate with glycolytic flux , are significantly higher in A - ( = 1 . 35 , nT - = 9 , nA - = 8 ) . Data information : Significance keys : * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 ( Welch ' s t - )

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(C) Concentrations of fructose-1,6-bisphosphate, which correlate with glycolytic flux, are significantly higher in A-strain (ratio = 1.35, nT-strain=9, nA-strain=8). Data information: Significance keys: * p<0.05, ** p<0.005, *** p<0.0005 (Welch's t-test)


( D ) Boxplots for selected metabolomics data indicate differences in energy redox status between T - A - strains ( nT - = 9 , nA - = 8 ) . Data information : Significance keys : * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 ( Welch ' s t - )

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(D) Boxplots for selected metabolomics data indicate differences in energy and redox status between T- and A-strains (nT-strain=9, nA-strain=8). Data information: Significance keys: * p<0.05, ** p<0.005, *** p<0.0005 (Welch's t-test)


( E ) of 432 genes that are differentially expressed at level between T - A - strains ( FDR < 10 % ) are measured in four conditions ( columns ) . First column : genes ordered by increasing fold - changes for RNAs ( computed as log2 [ T ] - log2 [ A ] ) . Second column : fold - changes for ( computed as log2 [ T ] - log2 [ A ] ) . Third column : fold - changes for RNAs in treated with rapamycin & caffeine ( TORC1 inhibition ; computed as log2 [ treatment ] - log2 [ control ] ) ( data from ref . ( Rallis et al , 2013 ) ) . Fourth column : fold - changes for RNAs in treated with H2O2 ( oxidative stress ; computed as log2 [ treatment ] - log2 [ control ] ) ( data from Chen et al , 2003 ) . Log2 fold - changes are capped at absolute values of 1 for columns .

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(E) Heatmap of the 432 genes that are differentially expressed at the RNA level between the T- and A-strains (FDR <10%) and are measured in all four conditions (columns). First column: genes ordered by increasing fold-changes for RNAs (computed as log2[T]-log2[A]). Second column: fold-changes for proteins (computed as log2[T]-log2[A]). Third column: fold-changes for RNAs in cells treated with rapamycin & caffeine (TORC1 inhibition; computed as log2[treatment]-log2[control]) (data from ref. (Rallis et al, 2013)). Fourth column: fold-changes for RNAs in cells treated with H2O2 (oxidative stress; computed as log2[treatment]-log2[control]) (data from Chen et al, 2003). Log2 fold-changes are capped at absolute values of 1 for all columns.


( A ) Growth measured by optical density ( OD ) at 600nm for 3 biological replicates each of T - A - strains over 6 hrs of exponential growth in YES media . x - axis refers to time since inoculation of culture . ( B ) same data as in ( A ) plotted on a log2 scale ( solid lines ) with lines fitted ( dotted lines ) . doubling time is inverse of slope .

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(A) Growth measured by optical density (OD) at 600nm for 3 biological replicates each of the T- and A-strains over 6 hrs of exponential growth in YES media. The x-axis refers to the time since inoculation of the culture. (B) The same data as in (A) plotted on a log2 scale (solid lines) with lines fitted (dotted lines). The doubling time is the inverse of the slope.


( C ) Media samples were taken from same cultures ( n = 3 for both strains ) at 8 hr timepoint remaining glucose was quantified . consumed glucose was calculated on amount of glucose measured in same , fresh media normalised to OD of each culture at time of sampling .

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(C) Media samples were taken from the same cultures (n=3 for both strains) at the 8 hr timepoint and the remaining glucose was quantified. The consumed glucose was calculated based on the amount of glucose measured in the same, fresh media and normalised to the OD of each culture at the time of sampling.


( D ) Oxygen consumption rates in A - T - strains ( of means = 0 . 65 , p = 0 . 0002 , Welch ' s T - , 3 biological replicates , each measured in technical duplicates ) . Data information : Vertical bars show mean of data . Error bars shaded areas in cases denote standard deviation . Significance keys : * p < 0 . 05 , * * * p < 0 . 0005 ( Welch ' s t - ) .

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(D) Oxygen consumption rates in A- and T-strains (ratio of means=0.65, p=0.0002, Welch's T-test, 3 biological replicates, each measured in technical duplicates). Data information: Vertical bars show the mean of the data. Error bars and shaded areas in all cases denote standard deviation. Significance keys: * p<0.05, *** p<0.0005 (Welch's t-test).


( E ) Culture dry after 24 hrs was measured reported as a fraction of of glucose put into media for 3 biological replicates per . Data information : Vertical bars show mean of data . Error bars shaded areas in cases denote standard deviation . Significance keys : * p < 0 . 05 , * * * p < 0 . 0005 ( Welch ' s t - ) .

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(E) Culture dry weight after 24 hrs was measured reported as a fraction of the weight of glucose put into the media for 3 biological replicates per strain. Data information: Vertical bars show the mean of the data. Error bars and shaded areas in all cases denote standard deviation. Significance keys: * p<0.05, *** p<0.0005 (Welch's t-test).


( A ) Spot assays on solid media from a three - fold dilution series of exponential cultures at same density in 96 - well plates ( 3 biological replicates of each ) spotted in 16 technical replicates ( each dilution in 4x4 square ) . A - is more resistant to antimycin A but less resistant to oxidative stress triggered by H2O2 or diamide . A control without toxin ( left ) was included in each batch of spot assays performed a representative image is shown here .

(A) Spot assays on solid media from a three-fold dilution series of exponential cultures at the same cell density in 96-well plates (3 biological replicates of each strain) and spotted in 16 technical replicates (each dilution in 4x4 square). The A-strain is more resistant to antimycin A but less resistant to oxidative stress triggered by H2O2 or diamide. A control without toxin (left) was included in each batch of spot assays performed and a representative image is shown here.


( B ) Fitness ( approximated by maximum slope of smoothed growth curves ) of A - T - strains on 12 carbon sources , with either yeast extract ( YE ) or ammonium ( NH4 ) , with or without 0 . 1 % priming glucose to support initial growth . For 48 conditions , two biological replicates of A - T - strains were grown in technical quadruplicates each . Dotted lines in panels mark a fitness of 1 ( i . e . , same fitness ) .

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(B) Fitness (approximated by maximum slope of smoothed growth curves) of A- and T-strains on 12 carbon sources, with either yeast extract (YE) or ammonium (NH4), with or without 0.1% priming glucose to support initial growth. For all 48 conditions, two biological replicates of A- and T-strains were grown in technical quadruplicates each. Dotted lines in panels mark a fitness ratio of 1 (i.e., same fitness).


( C ) Fitness of A - T - strains on 95 nitrogen sources on Biolog Phenotype MicroArrays . Conditions with no substantial growth were excluded ( black circles , maximum slope < 0 . 015 ) . Red lines show arbitrary significance cut - off , put at | log2 ( A - / T - ) | > 0 . 75 .

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(C) Fitness of A- and T-strains on 95 nitrogen sources on Biolog Phenotype MicroArrays. Conditions with no substantial growth were excluded (black circles, maximum slope <0.015). Red lines show arbitrary significance cut-off, put at |log2(A-strain/T-strain)|>0.75.


( D ) Fitness of A - T - strains on 72 different drugs toxins , at 4 concentrations each , on Biolog Phenotype MicroArrays . ( Results for benzamidine were inconclusive , with both strains comparatively resistant in one each . )

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(D) Fitness of A- and T-strains on 72 different drugs and toxins, at 4 concentrations each, on Biolog Phenotype MicroArrays. (Results for benzamidine were inconclusive, with both strains comparatively resistant in one concentration each.)


( E ) Spot assays to validate selected results Several assays were performed with both h - h90 strains , no mating - type specific differences were evident between two sets of allele - swap strains .

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(E) Spot assays to validate selected results Several assays were performed with both h- and h90 strains, and no mating-type specific differences were evident between the two sets of allele-swap strains.


( F ) Chronological lifespan of A - T - strains , i . e . proportion of non - dividing in stationary phase that maintain proliferative potential after refeeding . data show colony forming units ( CFUs ) per ml of culture over 7 days of stationary phase in glucose - depleted rich media . Three biological repeats were carried out for both strains , with each repeat measured as technical triplicates . Error bars represent standard error of biological replicates .

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(F) Chronological lifespan of A- and T-strains, i.e. the proportion of non-dividing cells in stationary phase that maintain proliferative potential after refeeding. The data show colony forming units (CFUs) per ml of culture over 7 days of stationary phase in glucose-depleted rich media. Three biological repeats were carried out for both strains, with each repeat measured as technical triplicates. Error bars represent standard error of the biological replicates.


( G ) Boxplot showing resistance to 3mM H2O2 grouped by pyk1 allele for 156 strains from our collection . T - strains had a higher mean fitness in H2O2 than A - strains ( 1 . 01 ± 0 . 06 vs 0 . 95 ± 0 . 11 ; p = 0 . 0021 , Welch ' s t - ) . resistance score was obtained as for antimycin A .

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(G) Boxplot showing resistance to 3mM H2O2 grouped by pyk1 allele for 156 strains from our collection. The T-strains had a higher mean fitness in H2O2 than the A-strains (1.01±0.06 vs 0.95±0.11; p=0.0021, Welch's t-test). The resistance score was obtained as for antimycin A.


( H ) Boxplot showing growth fitness on rich media , grouped by pyk1 allele for 158 S . pombe strains ( 0 . 88 ± 0 . 16 vs 0 . 94 ± 0 . 24 ; p = 0 . 18 , Welch ' s t - ) .

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(H) Boxplot showing growth fitness on rich media, grouped by pyk1 allele for 158 S. pombe strains (0.88±0.16 vs 0.94±0.24; p=0.18, Welch's t-test).


a , Starvation ( 3 h ) - induced autophagy results in a downregulation of H4K16ac in histone extracts of MEF .

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a, Starvation (3 h)-induced autophagy results in a downregulation of H4K16ac in histone extracts of MEF cells.


b , Upon rapamycin treatment ( 300 nM ) LC3 conversion downregulation of H4K16ac are observed in WT MEF but not in autophagy - deficient atg5 − / − atg7 − / − MEF .

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b, Upon rapamycin treatment (300 nM) LC3 conversion and downregulation of H4K16ac are observed in WT MEF cells but not in the autophagy-deficient atg5−/− and atg7−/− MEF cells.


c , Rapamycin treatment increased LC3 - II / LC3 - I promoted H4K16ac decrease in sirt1 − / − WT MEF .

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c, Rapamycin treatment increased the LC3-II/LC3-I ratio and promoted H4K16ac decrease in sirt1−/− and WT MEF cells.


d , Rapamycin - induced autophagy led to downregulation of H4K16ac at 48 h in histone extracts of HeLa U20S , after 6 h in U1810 .

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d, Rapamycin-induced autophagy led to downregulation of H4K16ac at 48 h in histone extracts of HeLa and U20S cells, and after 6 h in U1810 cells.


e , of H4K16 acetylation by immunoblotting is depicted for rapamycin - treated . Data are expressed as mean ± s . e . m . ( n = 3 - 5 ) ; * P 0 . 05 ; * * P 0 . 01 .

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e, Quantification of H4K16 acetylation by immunoblotting is depicted for rapamycin-treated cells. Data are expressed as mean ± s.e.m. (n = 3-5); *P 0.05; **P 0.01.


Rapamycin treatment ( 48 h ) promoted downregulation of H4K16 histone acetyltransferase hMOF expression level in MEF ( a )

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Rapamycin treatment (48 h) promoted the downregulation of the H4K16 histone acetyltransferase hMOF expression level in MEF (a)


Rapamycin treatment ( 48 h ) promoted downregulation of H4K16 histone acetyltransferase hMOF expression level in transfected HeLa ( b ) .

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Rapamycin treatment (48 h) promoted the downregulation of the H4K16 histone acetyltransferase hMOF expression level in transfected HeLa cells (b).


VPA ( 1 mM ) treatment counteracted rapamycin - induced H4K16ac downregulation ( c ) decreased LC3 ( d ) .

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VPA (1 mM) treatment counteracted rapamycin-induced H4K16ac downregulation (c) and decreased the LC3 ratio (d).


e , Co - treatment with chloroquine ( CQ , 10 µM ) showed that decrease in LC3 was a result of an increase in autophagic flux .

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e, Co-treatment with chloroquine (CQ, 10 µM) showed that the decrease in LC3 ratio was a result of an increase in autophagic flux.


f , Inhibition of autophagy by CQ after hMOF overexpression shows that hMOF does not inhibit autophagic flux .

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f, Inhibition of autophagy by CQ after hMOF overexpression shows that hMOF does not inhibit autophagic flux.


g , yeast homologue of hMOF , Sas2 , tagged with 3 × HA showed a complete disappearance of HA signal upon autophagy induction after 3 h .

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g, The yeast homologue of hMOF, Sas2, tagged with 3× HA showed a complete disappearance of HA signal upon autophagy induction after 3 h.


h , Overexpression of Sas2 repressed downregulation of H4K16ac upon rapamycin treatment .

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h, Overexpression of Sas2 repressed the downregulation of H4K16ac upon rapamycin treatment.


a , VPA increases autophagic flux in rapamycin - treated HeLa transfected with mRFP - GFP - LC3 tandem reporter construct which allows distinction between autophagosomes ( GFP + / RFP + yellow ) autolysosomes ( GFP − / RFP + red ) .

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a, VPA increases the autophagic flux in rapamycin-treated HeLa cells transfected with the mRFP-GFP-LC3 tandem reporter construct which allows distinction between autophagosomes (GFP+/RFP+ yellow puncta) and autolysosomes (GFP−/RFP+ red puncta).


b , Confocal microscopy image of a treated with rapamycin VPA depicting a high of red to green LC3 indicating an increase in autophagic flux .

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b, Confocal microscopy image of a cell treated with rapamycin and VPA depicting a high ratio of red to green LC3 puncta indicating an increase in autophagic flux.


c , Co - treatment with VPA rapamycin led to increased .

c, Co-treatment with VPA and rapamycin led to increased cell death.


d , Co - treatment with CQ abrogated VPA + rapamycin - induced .

d, Co-treatment with CQ abrogated VPA + rapamycin-induced cell death.


e , f , Increasing H4K16ac by either overexpression of hMOF , inhibition of SIRT1 by siRNA or chemical inhibitor Ex527 promoted upon rapamycin treatment .

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e, f, Increasing H4K16ac levels by either overexpression of hMOF, inhibition of SIRT1 by siRNA or the chemical inhibitor Ex527 promoted cell death upon rapamycin treatment.


Co - treatment with autophagy inhibitor bafilomycin A1 ( BafA , 40 nM ) ( g ) or 3 - methyladenine ( 3MA , 5 mM ) ( h ) abrogated VPA + rapamycin - induced .

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Co-treatment with the autophagy inhibitor bafilomycin A1 (BafA, 40 nM) (g) or 3-methyladenine (3MA, 5 mM) (h) abrogated VPA + rapamycin-induced cell death.


Treatment of HeLa upon - acid starvation with VPA induced ( i ) , which was rescued when were co - treated with CQ ( j ) . Data are expressed as mean ± s . e . m . ( n = 3 ) .

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Treatment of HeLa cells upon amino-acid starvation with VPA induced cell death (i), which was rescued when cells were co-treated with CQ (j). Data are expressed as mean ± s.e.m. (n = 3).


A sWGS performed on sorted CD3 + / Vβ + malignant T - isolated from blood of P1 , shown as a representative example . Copy number profiles ploidy estimates were calculated using ichorCNA . Blue represents balanced genomic regions , green indicates loss / µdeletion , red indicates gain / amplification of regions on chromosome 17 . Darker color shades indicate higher log - 2 ratios .

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A sWGS performed on sorted CD3+/Vβ+ malignant T-cells isolated from the blood of P1, shown as a representative example. Copy number profiles and ploidy estimates were calculated using ichorCNA. Blue represents balanced genomic regions, green indicates loss/µdeletion, and red indicates gain/amplification of regions on chromosome 17. Darker color shades indicate higher log-2 ratios.


B of extracted CNA [ log2 ratios ] of STAT genes on chr17 ( i . e . , STAT3 , STAT5A STAT5B ) , other relevant suppressors ( i . e , STAT1 , SOCS1 TP53 ) oncogenes ( MYC ) . Red indicates gain / amplification , blue indicates loss / deletion . balance threshold was set to + / - 0 . 1 . P33 presented with two malignant clones in blood both were sequenced ( P33 _ 1 ( Clone 1 ) , P33 _ 2 ( Clone 2 ) ) . * Tetraploid malignant clone .

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B Heatmap of extracted CNA [log2 ratios] of STAT genes on chr17 (i.e., STAT3, STAT5A and STAT5B), and other relevant tumor suppressors (i.e, STAT1, SOCS1 and TP53) and oncogenes (MYC). Red indicates gain/amplification, blue indicates loss/deletion. The balance threshold was set to +/- 0.1. P33 presented with two malignant clones in the blood and both were sequenced (P33_1 (Clone 1), P33_2 (Clone 2)). *Tetraploid malignant clone.


A - C Spearman correlation using CNA log2 ratios of clonal CD3 + detected in for ( A ) STAT3 , ( B ) STAT5A ( C ) STAT5B .

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A-C Spearman correlation analysis using CNA log2 ratios and the percentage of clonal CD3+ cells detected in patients for (A) STAT3, (B) STAT5A and (C) STAT5B.


D - F RT - qPCR expression data of ( C ) STAT3 , ( D ) STAT5A , ( E ) STAT5B genes from whole PBMCs isolated from blood ( n = 5 ) or healthy controls ( n = 9 ) . GAPDH expression was used for normalization . Statistical significance was calculated using two - unpaired t - . P - value : < 0 . 01 ( * * ) , < 0 . 0001 ( * * * * ) . One experiment was performed in technical triplicates . bold dashed in middle of violin plot denotes median value , while thin dotted lines denote interquartile range .

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D-F RT-qPCR expression data of (C) STAT3, (D) STAT5A, and (E) STAT5B genes from whole PBMCs isolated from patient blood (n = 5) or healthy controls (n = 9). GAPDH expression was used for normalization. Statistical significance was calculated using two-tailed unpaired t-test. P-value: <0.01 (**), <0.0001 (****). One experiment was performed in technical triplicates. The bold dashed line in the middle of the violin plot denotes the median value, while the thin dotted lines denote the interquartile range.


G Immunoblotting for total STAT3 / 5 isolated from respective whole PBMCs from blood ( n = 3 ) , or from healthy controls ( n = 3 ) . HSC70 was used as loading control . One experiment was performed .

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G Immunoblotting for total STAT3/5 protein isolated from the respective whole PBMCs from patient blood (n = 3), or from healthy controls (n = 3). HSC70 was used as loading control. One experiment was performed.


H Representative image of H & E , CD3 , pY - STAT5 single CD3 / pY - STAT5 double on skin biopsy tissue from P3 ( n = 1 ) , which demonstrates infiltration of CD3 + pY - STAT5 + ( 13 . 45 % ) into dermal subcutaneous layer of skin . Arrows indicate pY - STAT5 + single CD3 + pY - STAT5 + .

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H Representative image of H&E, CD3, pY-STAT5 single staining and CD3/pY-STAT5 double staining on skin biopsy tissue from P3 (n = 1), which demonstrates infiltration of CD3+ pY-STAT5+ (13.45%) cells into the dermal and subcutaneous layer of the skin. Arrows indicate pY-STAT5+ single and CD3+ pY-STAT5+ cells.


I - M of from skin - biopsy sections of L - CTCL ( n = 6 ) healthy individuals ( n = 5 ) As number of STAT5A / B positive was negligible ( less than 0 . 03 / per mm2 ) in healthy skin biopsy sections , healthy control samples were considered negative for pY - STAT5 , were set to 0 . Statistical significance was calculated using an unpaired Welch ' s t - . P - value : < 0 . 05 ( * ) . bold dashed in middle of violin plot denotes median value , while thin dotted lines denote interquartile range .

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I-M Quantification of IHC staining from skin-biopsy sections of L-CTCL patients (n = 6) and healthy individuals (n = 5) As the number of STAT5A/B positive cells was negligible (less than 0.03/per mm2) in healthy skin biopsy sections, healthy control samples were considered negative for pY-STAT5, and levels were set to 0. Statistical significance was calculated using an unpaired Welch's t-test. P-value: <0.05 (*).The bold dashed line in the middle of the violin plot denotes the median value, while the thin dotted lines denote the interquartile range.


A - C Graphic depicts aberration patterns on ( A ) chromosome 17 ( STAT3 / 5 ) , ( B ) chromosome 2 ( STAT1 ) ( C ) chromosome 16 ( SOCS1 ) in CTCL lines used in study . Aberration patterns are represented as log2 of of vs . reference were generated by aCGH . Immunoblotting for total STAT1 / 3 / 5 activation : phospo - Tyr ( 701 ) - STAT1 / phospho - Tyr ( 705 ) - STAT3 / phospho - Tyr ( 694 / 699 ) - STAT5 , SOCS1 . cytokine - dependent , SeAx , was collected 30 min after 5 ng / ml IL - 2 IL - 4 cytokine addition . HSC70 was used as loading control . One representative out of three independent experiments is shown .

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A-C Graphic depicts the aberration patterns on (A) chromosome 17 (STAT3/5), (B) chromosome 2 (STAT1) and (C) chromosome 16 (SOCS1) in the CTCL cell lines used in the study. Aberration patterns are represented as the log2 ratio of the fluorescence intensity of the tumor DNA vs. reference DNA and were generated by aCGH. Immunoblotting for total protein STAT1/3/5 and activation levels: phospo-Tyr (701)-STAT1/phospho-Tyr (705)-STAT3/phospho-Tyr (694/699)-STAT5, and SOCS1 protein levels. The cytokine-dependent cell line, SeAx, was collected 30 min after 5 ng/ml IL-2 and IL-4 cytokine addition. HSC70 was used as loading control. One representative out of three independent experiments is shown.


A of IC50 values calculated from drug response using CellTiter - Glo viability assays upon 48h drug treatment . One representative , out of three independent experiments performed in triplicates , is shown .

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A Heatmap of IC50 values calculated from drug response analysis using CellTiter-Glo viability assays upon 48h drug treatment. One representative, out of three independent experiments performed in triplicates, is shown.


B , Immunoblot showing total STAT3 / 5 activation : phospho - Tyr ( 705 ) - STAT3 / phospho - Tyr ( 694 / 699 ) - STAT5A / B after 24h treatment with ( B ) JPX - 0750 cytokine - dependent , SeAx , was collected 30 min after 5 ng / ml IL - 2 IL - 4 cytokine addition . HSC70 was used as loading control . normalized of phospho - total STAT3 / 5 quantified by densitometry , are shown below respective blots . One representative , out of three independent experiments is shown .

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B, Immunoblot showing total protein STAT3/5 and activation levels: phospho-Tyr (705)-STAT3/phospho-Tyr (694/699)-STAT5A/B after 24h treatment with (B) JPX-0750 The cytokine-dependent cell line, SeAx, was collected 30 min after 5 ng/ml IL-2 and IL-4 cytokine addition. HSC70 was used as loading control. The normalized levels of phospho- and total STAT3/5 quantified by densitometry, are shown below the respective blots. One representative, out of three independent experiments is shown.


C Immunoblot showing total STAT3 / 5 activation : phospho - Tyr ( 705 ) - STAT3 / phospho - Tyr ( 694 / 699 ) - STAT5A / B after 24h treatment with ( C ) IQDMA . cytokine - dependent , SeAx , was collected 30 min after 5 ng / ml IL - 2 IL - 4 cytokine addition . HSC70 was used as loading control . normalized of phospho - total STAT3 / 5 quantified by densitometry , are shown below respective blots . One representative , out of three independent experiments is shown .

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C Immunoblot showing total protein STAT3/5 and activation levels: phospho-Tyr (705)-STAT3/phospho-Tyr (694/699)-STAT5A/B after 24h treatment with (C) IQDMA. The cytokine-dependent cell line, SeAx, was collected 30 min after 5 ng/ml IL-2 and IL-4 cytokine addition. HSC70 was used as loading control. The normalized levels of phospho- and total STAT3/5 quantified by densitometry, are shown below the respective blots. One representative, out of three independent experiments is shown.


Real - Time apoptosis / necrosis in ( D , SeAx ( F , Myla lines treated with 2 . 5µM JPX - 0750 RLU , Apoptosis [ RLU ] = relative luminescence ; Necrosis [ RFU ] = relative units . Error bars represent mean + / - SD . One experiment in technical triplicates was performed .

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Real-Time apoptosis/necrosis assay in the (D, SeAx and (F, Myla cell lines treated with 2.5µM JPX-0750 RLU, Apoptosis [RLU] = relative luminescence; Necrosis [RFU] = relative fluorescence units. Error bars represent mean +/-SD. One experiment in technical triplicates was performed.


Real - Time apoptosis / necrosis in E ) SeAx G ) Myla lines treated with 2 . 5µM IQDMA . RLU , Apoptosis [ RLU ] = relative luminescence ; Necrosis [ RFU ] = relative units . Error bars represent mean + / - SD . One experiment in technical triplicates was performed .

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Real-Time apoptosis/necrosis assay in the E) SeAx and G) Myla cell lines treated with 2.5µM IQDMA. RLU, Apoptosis [RLU] = relative luminescence; Necrosis [RFU] = relative fluorescence units. Error bars represent mean +/-SD. One experiment in technical triplicates was performed.


H - I effect of ( H ) JPX - 0750 ( I ) IQDMA on viability of CTCL in comparison to viability of juvenile fibroblasts ( JF ) keratinocytes ( HaCat ) at a low dose range ( 0 . 7 - 3µM ) . One experiment , in triplicates , was performed with JF HaCat , three experiments , in triplicates with CTCL . Error bars represent mean + / - SD . Statistical significance was calculated by two - way ANOVA with Tukey ' s multiple comparisons . P - value : < 0 . 05 ( * ) , < 0 . 01 ( * * ) , < 0 . 001 ( * * * ) , < 0 . 0001 ( * * * * ) .

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H-I The effect of (H) JPX-0750 and (I) IQDMA on the viability of CTCL cells in comparison to viability of primary juvenile fibroblasts (JF) and keratinocytes (HaCat) at a low dose range (0.7-3µM). One experiment, in triplicates, was performed with JF and HaCat cells, and three experiments, in triplicates with the CTCL cells. Error bars represent mean +/-SD. Statistical significance was calculated by two-way ANOVA with Tukey's multiple comparisons test. P-value: <0.05 (*), <0.01 (**), <0.001 (***), <0.0001 (****).


B Subcellular fractions of SeAx treated with IQDMA for 24h immunoblotted for pY - STAT5 total STAT5 . were collected 30 min after 1 ng / ml IL - 2 cytokine addition . α - Tubulin Histone H3 were used as loading controls for fractions , respectively . normalized of phospho - total STAT5 in nucleus cytoplasm , quantified by densitometry , are shown below respective blots . One representative out of two independent experiments is shown .

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B Subcellular fractions of SeAx cells treated with IQDMA for 24h and immunoblotted for pY-STAT5 and total STAT5. The cells were collected 30 min after 1 ng/ml IL-2 cytokine addition. α-Tubulin and Histone H3 were used as loading controls for cytoplasmic and nuclear fractions, respectively. The normalized levels of phospho- and total STAT5 in the nucleus and cytoplasm, quantified by densitometry, are shown below the respective blots. One representative out of two independent experiments is shown.


D - F Scatter plot depicting profile of SeAx treated with ( D ) 1µM IQDMA , ( E ) 2 . 5µM IQDMA ( F ) 10µM IQDMA . abundances were determined using TMT - mass spectrometry Significant changes were assessed by a modified t - as implemented in limma package , with negative log10 p values on x - axis , log2 fold change shown on y - axis .

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D-F Scatter plot depicting the profile of SeAx cells treated with (D) 1µM IQDMA, (E) 2.5µM IQDMA and (F) 10µM IQDMA. Protein abundances were determined using TMT-based quantification mass spectrometry Significant changes were assessed by a modified t-test as implemented in the limma package, with the negative log10 p values on the x-axis, and log2 fold change shown on the y-axis.


G of IC50 values calculated from drug response using CellTiter - Blue or CellTiter - Glo viability assays upon 48h drug treatment . One representative out of three independent experiments performed in triplicates is shown . Grey represents presence of STAT3 / 5 gain .

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G Heatmap of IC50 values calculated from drug response analysis using CellTiter-Blue or CellTiter-Glo viability assays upon 48h drug treatment. One representative out of three independent experiments performed in triplicates is shown. Grey represents the presence of STAT3/5 gain.


A Inhibitor treatment was carried out with increased dose escalation for 24h . SeAx were collected 2h after 7 , 5 ng / ml IL - 2 IL - 4 cytokine addition . STAT3 / 5 target gene products MYC , PIM1 , MCL - 1 , AKT1 / 2 / 3 , as well as regulating cycle , such as CYCLIN D2 D3 , CDK6 were probed by Western blotting . HSC70 served as loading control . normalized , quantified by densitometry , are shown below respective blots . One representative out of three independent experiments is shown .

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A Inhibitor treatment was carried out with increased dose escalation for 24h. SeAx cells were collected 2h after 7,5 ng/ml IL-2 and IL-4 cytokine addition. STAT3/5 target gene products MYC, PIM1, MCL-1, and AKT1/2/3, as well as proteins regulating the cell cycle, such as CYCLIN D2 and D3, and CDK6 were probed by Western blotting. HSC70 served as loading control. The normalized protein levels, quantified by densitometry, are shown below the respective blots. One representative out of three independent experiments is shown.


Synergy of indicated two - drug combinations in SeAx Myla after 48 h treatment . In each graph most synergistic ( MSA ) is highlighted , which represents most synergistic 3 - by - 3 dose - window with respective MSA score . Zero interaction potency model was applied to quantify degree of synergy , according to which an MSA score below - 10 indicates that drugs are antagonistic ( green ) , a score between - 10 10 indicates that 2 drugs are additive ( ) , while a score above 10 indicates a synergistic effect ( red ) .

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Synergy analysis of the indicated two-drug combinations in the SeAx and Myla cells after 48 h treatment. In each graph the most synergistic area (MSA) is highlighted, which represents the most synergistic 3-by-3 dose-window with the respective MSA score. The Zero interaction potency model was applied to quantify the degree of synergy, according to which an MSA score below -10 indicates that drugs are antagonistic (green), a score between -10 and 10 indicates that 2 drugs are additive (white), while a score above 10 indicates a synergistic effect (red).


( D ) showing MSA scores of combination of FRAx597 with listed drugs . One representative out of two independent experiments is shown .

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(D) Heatmap showing MSA scores of the combination of FRAx597 with the listed drugs. One representative out of two independent experiments is shown.


E showing IC50 values upon treatment of PBMCs isolated from L - CTCL with JPX - 0750 , IQDMA FRAx597 . One experiment performed in triplicates using CellTiter - Glo viability assays upon 48h drug treatment is shown . relative STAT3 / 5 log2 value is depicted in grey .

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E Heatmap showing the IC50 values upon treatment of primary PBMCs isolated from L-CTCL patients with JPX-0750, IQDMA and FRAx597. One experiment performed in triplicates using CellTiter-Glo viability assays upon 48h drug treatment is shown. The relative STAT3/5 log2 ratio value is depicted in grey.


F - H Spearman correlation using CNA log2 ratios IC50 values for respective drug .

F-H Spearman correlation analysis using CNA log2 ratios and the IC50 values for the respective drug.


B Growth rate of Hut78 , Myla H9 - derived tumors measured on respective day post - injection . mean of respective volumes for H9 group for vehicle - treated groups for Hut78 Myla are shown . volume = ( x width2 ) / 2 . Error bars represent mean + / - . n represents total number of tumors that engrafted per group .

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B Growth rate of Hut78, Myla and H9-derived tumors measured on the respective day post-injection. The mean of the respective tumor volumes for H9 group and for the vehicle-treated groups for Hut78 and Myla are shown. Tumor volume = (length x width2)/2. Error bars represent mean +/-SEM. n represents the total number of tumors that engrafted per group.


C - D Myla - derived upon experiment termination , after 20 days of treatment with 5 mg / kg of ( C ) JPX - 0750 ( n = 5 ) IQDMA ( n = 6 ) , or vehicle ( n = 7 ) , ( D ) FRAx597 ( n = 5 ) or vehicle ( n = 4 ) , with n representing number of analyzed tumors per group . Statistical significance was calculated using two - paired t - . P - value : < 0 . 05 ( * ) . Error bars represent mean + / - .

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C-D Myla-derived tumor weight upon experiment termination, after 20 days of treatment with 5 mg/kg of (C) JPX-0750 (n = 5) and IQDMA (n = 6), or vehicle (n = 7), and (D) FRAx597 (n = 5) or vehicle (n = 4), with n representing the number of analyzed tumors per group. Statistical significance was calculated using two-tailed paired t-test. P- value: <0.05 (*). Error bars represent mean +/-SEM.


H & E of Myla - derived tumors treated with JPX - 0750 ( n = 6 ) , IQDMA ( n = 6 ) or vehicle ( n = 6 ) , FRAx597 ( n = 5 ) or vehicle ( n = 4 ) , stained with ( E , F ) H & E with n representing number of analyzed tumors per group . Pictures shown are from contiguous sections . Dotted rectangles indicate magnified areas . Scale bars , 200 , 100 25 µm . Violin plots show perimeter of annotated - infiltration / expansion region into intradermal subcutaneous region of skin Statistical significance was calculated using two - paired t - with Welch ´ s correction . P - value : < 0 . 05 ( * ) , < 0 . 01 ( * * ) , < 0 . 001 ( * * * ) . bold dashed in middle of violin plot denotes median value , while thin dotted lines denote interquartile range .

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H&E analysis of Myla-derived tumors treated with JPX-0750 (n = 6), IQDMA (n = 6) or vehicle (n = 6), and FRAx597 (n = 5) or vehicle (n = 4), stained with (E, F) H&E with n representing the number of analyzed tumors per group. Pictures shown are from contiguous sections. Dotted rectangles indicate magnified areas. Scale bars, 200, 100 and 25 µm. Violin plots show the perimeter of the annotated tumor cell-infiltration/expansion region into the intradermal and subcutaneous region of the skin Statistical significance was calculated using two-tailed paired t-test with Welch´s correction. P-value: <0.05 (*), <0.01 (**), <0.001 (***). The bold dashed line in the middle of the violin plot denotes the median value, while the thin dotted lines denote the interquartile range.


of Myla - derived tumors treated with JPX - 0750 ( n = 6 ) , IQDMA ( n = 6 ) or vehicle ( n = 6 ) , FRAx597 ( n = 5 ) or vehicle ( n = 4 ) , stained with ( G , H ) Ki67 , as a marker for proliferation with n representing number of analyzed tumors per group . Pictures shown are from contiguous sections . Dotted rectangles indicate magnified areas . Scale bars , 200 , 100 25 µm . Violin plots show of of Ki67 + in tissue . Statistical significance was calculated using two - paired t - with Welch ´ s correction . P - value : < 0 . 05 ( * ) , < 0 . 01 ( * * ) , < 0 . 001 ( * * * ) . bold dashed in middle of violin plot denotes median value , while thin dotted lines denote interquartile range .

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IHC analysis of Myla-derived tumors treated with JPX-0750 (n = 6), IQDMA (n = 6) or vehicle (n = 6), and FRAx597 (n = 5) or vehicle (n = 4), stained with (G, H) Ki67, as a marker for proliferation with n representing the number of analyzed tumors per group. Pictures shown are from contiguous sections. Dotted rectangles indicate magnified areas. Scale bars, 200, 100 and 25 µm. Violin plots show the quantification of the percentage of Ki67+ cells in the tissue. Statistical significance was calculated using two-tailed paired t-test with Welch´s correction. P-value: <0.05 (*), <0.01 (**), <0.001 (***). The bold dashed line in the middle of the violin plot denotes the median value, while the thin dotted lines denote the interquartile range.


of Myla - derived tumors treated with JPX - 0750 ( n = 6 ) , IQDMA ( n = 6 ) or vehicle ( n = 6 ) , FRAx597 ( n = 5 ) or vehicle ( n = 4 ) , stained with ( I , J ) CD31 , as a vessel marker , with n representing number of analyzed tumors per group . Pictures shown are from contiguous sections . Dotted rectangles indicate magnified areas . Scale bars , 200 , 100 25 µm . CD31 was quantified as raw of vessels with lumen . Statistical significance was calculated using two - paired t - with Welch ´ s correction . P - value : < 0 . 05 ( * ) , < 0 . 01 ( * * ) , < 0 . 001 ( * * * ) . bold dashed in middle of violin plot denotes median value , while thin dotted lines denote interquartile range .

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IHC analysis of Myla-derived tumors treated with JPX-0750 (n = 6), IQDMA (n = 6) or vehicle (n = 6), and FRAx597 (n = 5) or vehicle (n = 4), stained with (I, J) CD31, as a tumor vessel marker, with n representing the number of analyzed tumors per group. Pictures shown are from contiguous sections. Dotted rectangles indicate magnified areas. Scale bars, 200, 100 and 25 µm. CD31 staining was quantified as raw counts of vessels with lumen. Statistical significance was calculated using two-tailed paired t-test with Welch´s correction. P-value: <0.05 (*), <0.01 (**), <0.001 (***). The bold dashed line in the middle of the violin plot denotes the median value, while the thin dotted lines denote the interquartile range.


( B ) Histogram displaying SILAC ratios ( log2 ) of stress - induced phosphorylation sites in setup cdc55Δ ( upper panel ) setup rts1Δ ( lower panel ) ( Hollenstein et al . , 2020 ) , stratified by their Hog1 - dependence ( grey versus thick black ) .

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(B) Histogram displaying the SILAC ratios (log2) of stress-induced phosphorylation sites in setup cdc55Δ (upper panel) and setup rts1Δ (lower panel) (Hollenstein et al., 2020), stratified by their Hog1-dependence (grey area versus thick black line).


( D ) plot monitoring growth rate ( y - axis ) of indicated strains ( colored lines ) under different osmolarity conditions ( x - axis ) .

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(D) Line plot monitoring growth rate (y-axis) of indicated strains (colored lines) under different osmolarity conditions (x-axis).


B ) Western - blot illustrating depletion of AID - Cdc55 at various time points after addition of auxin ( upper panel ) ; Pgk2 serves as a negative control ( lower panel ) .

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B) Western-blot illustrating the depletion of AID-Cdc55 at various time points after addition of auxin (upper panel); Pgk2 serves as a negative control (lower panel).


( D ) Transcriptional of stress - induced expression of ESR genes CTT1 ( upper panel ) PGM2 ( lower panel ) under different conditions ( i . e . presence / absence of IGO1 / 2 induction of iAID - Cdc55 depletion ) . Measured time points after stress application are depicted on x - axis , target gene mRNA are illustrated on y - axis as fold over reference gene IPP1 , values are normalized relative to wild type without induction of iAID - Cdc55 depletion at 30 minutes post - stress .

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(D) Transcriptional analysis of stress-induced expression of ESR genes CTT1 (upper panel) and PGM2 (lower panel) under different strain conditions (i.e. presence/absence of IGO1/2 and induction of iAID-Cdc55 depletion). Measured time points after stress application are depicted on the x-axis, target gene mRNA levels are illustrated on the y-axis as fold over reference gene IPP1, values are normalized relative to wild type without induction of iAID-Cdc55 depletion at 30 minutes post-stress.


E ) Transcriptional of stress - induced expression of ESR genes CTT1 ( upper panel ) PGM2 ( lower panel ) at 30 minutes post - stress . Target gene mRNA are illustrated on y - axis as fold over reference gene IPP1 , values are normalized relative to wild type without induction of iAID - Cdc55 depletion . dep . : Cdc55 depleted , n . d . : Cdc55 non - depleted .

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E) Transcriptional analysis of stress-induced expression of ESR genes CTT1 (upper panel) and PGM2 (lower panel) at 30 minutes post-stress. Target gene mRNA levels are illustrated on the y-axis as fold over reference gene IPP1, values are normalized relative to wild type without induction of iAID-Cdc55 depletion. dep.: Cdc55 depleted, n.d.: Cdc55 non-depleted.


( C ) Mobility shift assays monitoring phosphorylation - induced mobility changes of Gis1 ( two upper rows ) Rph1 ( two lower rows ) upon hyperosmotic stress in wild type , igo1 ∆ igo2 ∆ Cdc55 - depleted ( right - side ) .

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(C) Mobility shift assays monitoring phosphorylation-induced mobility changes of Gis1 (two upper rows) and Rph1 (two lower rows) upon hyperosmotic stress in wild type, igo1∆igo2∆ and Cdc55-depleted cells (right-hand side).


( E ) Western blot phosphorylation shift assays of wild type Gis1 - myc ( upper panel ) Rph1 - Flag ( lower panel ) , mutated forms Gis1 - 5A - myc ( upper panel ) Rph1 - 5A - Flag ( lower panel ) . measurement was done using Phos - tag ( Kinoshita et al . , 2006 ) at indicated time points after exposure to hyperosmotic stress ( 0 . 5 M NaCl ) using Pgk1 as a negative control . location of mutated residues is shown in respective schematics . Red boxes : demethylase associated domains ( JmjN JmjC ) , blue box : zinc finger domain .

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(E) Western blot phosphorylation shift assays of wild type Gis1-myc (upper panel) and Rph1-Flag (lower panel), and mutated forms Gis1-5A-myc (upper panel) and Rph1-5A-Flag (lower panel). The measurement was done using Phos-tag (Kinoshita et al., 2006) and at the indicated time points after exposure to hyperosmotic stress (0.5 M NaCl) using Pgk1 protein levels as a negative control. The location of the mutated residues is shown in the respective schematics. Red boxes: demethylase associated domains (JmjN and JmjC), blue box: zinc finger domain.