Figure 2 ( 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 + 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 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 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 .

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

Figure 2(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+ 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 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 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.


Figure 3 ( 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 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 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 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 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 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 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 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 ) .

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

Figure 3(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 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 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 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 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 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 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 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).


Figure 4 ( 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 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 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 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 .

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

Figure 4(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 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 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 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.


Figure 1Tissue microarrays ( TMAs ) from primary PCa ( n = 132 ) and CRPC ( n = 148 ) were stained via immunohistochemistry for poly ( ADP - ribose ) ( PAR ) , and scored by a clinical pathologist ( T . Parsons ) for intensity ( 0 - 3 ) and percentage ( 0 - 3 ) PAR score was generated via the equation : ( intensity x 1 ) + ( percentage x 2 ) . PAR scores were compared between primary and CRPC . * * * * = p value < 0 . 0001 by Chi - square testManual PAR scores were divided in to quartiles and then were compared to progression - free survival in the CRPC TMAs . * = p < 0 . 05 , ns = not statistically significant by Log - rank ( Mantel - Cox ) Representative image of one TMA core after multiplex fluorescent IHC for γH2AX ( green ) , PAR ( red ) , PARP - 1 ( purple ) , with DNA ( blue ) . Top Right : Insets of parent image at left . Numbers above inset columns coincide with numbers on image at left that were chosen for further magnification and representation ( boxed areas ) . Bottom Left : Percent positive staining for PAR for the entirety of each TMA cohort . Bottom Middle : Percent positive staining for PARP - 1 . Bottom right : γH2AX for the entirety of each TMA cohort . Data was considered after a median intensity cutoff , and analyzed for statistical significance using two - tailed Student " s t - test for PAR , PARP - 1 , and γH2AX , respectively . Exact p values are indiTwo - tailed Spearman correlation test between PAR and γH2AX ( % positive with a median intensity cut - off ) . Exact p values are indicated when

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

Figure 1Tissue microarrays (TMAs) from primary PCa (n=132) and CRPC (n=148) were stained via immunohistochemistry for poly(ADP-ribose) (PAR), and scored by a clinical pathologist (T. Parsons) for intensity (0-3) and percentage (0-3) PAR score was generated via the equation: (intensity x 1) + (percentage x 2). PAR scores were compared between primary and CRPC. ****=p value<0.0001 by Chi-square testManual PAR scores were divided in to quartiles and then were compared to progression-free survival in the CRPC TMAs. *=p<0.05, ns=not statistically significant by Log-rank (Mantel-Cox)Representative image of one TMA core after multiplex fluorescent IHC for γH2AX (green), PAR (red), PARP-1 (purple), with DNA (blue). Top Right: Insets of parent image at left. Numbers above inset columns coincide with numbers on image at left that were chosen for further magnification and representation (boxed areas). Bottom Left: Percent positive staining for PAR for the entirety of each TMA cohort. Bottom Middle: Percent positive staining for PARP-1. Bottom right: γH2AX for the entirety of each TMA cohort. Data was considered after a median intensity cutoff, and analyzed for statistical significance using two-tailed Student"s t-test for PAR, PARP-1, and γH2AX, respectively. Exact p values are indiTwo-tailed Spearman correlation test between PAR and γH2AX (% positive with a median intensity cut-off). Exact p values are indicated when available


Figure 2Top , left : Schematic representing the conditions utilized for transcriptomic analyses ( n = 2 ) of HT - sensitive LNCaP cells . Cells were deprived of hormones for 72 hours , followed by either treatment with 2 . 5uM veliparib ( PARPi ) or vehicle control ( DMSO ) for 1 hour , then subsequently treated with either 1nM DHT or vehicle control ( EtOH ) for 16 hours . Top , right : Immunoblot with the indicated antisera . Bottom : Volcano plots of transcripts found to be differentially regulated by DHT v . EtOH ( left ) or DHT v . PARPi followed by DHT ( right ) . Red dots indicate transcripts that were both statistically significantly altered ( p < 0 . 05 ) and more than 1 . 5 - fold changedTop , left : Schematic representing the conditions utilized for transcriptomic analyses ( n = 2 ) of CRPC C4 - 2 cells . Cells were deprived of hormones for 72 hours , followed by either treatment with 2 . 5uM veliparib ( PARPi ) or vehicle control ( DMSO ) for for 16 hours . Top , right : Immunoblot with the indicated antisera . Bottom : Volcano plots of transcripts found to be differentially regulated PARPi v . vehicle control . Red dots indicate transcripts that were both statistically significantly altered ( p < 0 . 05 ) and more than 1 . 5 - fold changedGenes found to be down - regulated by PARPi as described above ( p value < 0 . 05 , 1 . 5 - fold change ) in either HT - sensitive cells ( left ) or CRPC cells ( right ) were queried against the expression of these genes in the Grasso et al data set in Oncomine . Benign = grey , primary PCa = blue , metastases = orange . Boxplot was generated using the mean expression of the PARPi down - regulated genes in the indicated data sets . Statistical significance determined by two - tailed Student " s

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Figure 2Top, left: Schematic representing the conditions utilized for transcriptomic analyses (n=2) of HT-sensitive LNCaP cells. Cells were deprived of hormones for 72 hours, followed by either treatment with 2.5uM veliparib (PARPi) or vehicle control (DMSO) for 1 hour, then subsequently treated with either 1nM DHT or vehicle control (EtOH) for 16 hours. Top, right: Immunoblot with the indicated antisera. Bottom: Volcano plots of transcripts found to be differentially regulated by DHT v. EtOH (left) or DHT v. PARPi followed by DHT (right). Red dots indicate transcripts that were both statistically significantly altered (p<0.05) and more than 1.5-fold changedTop, left: Schematic representing the conditions utilized for transcriptomic analyses (n=2) of CRPC C4-2 cells. Cells were deprived of hormones for 72 hours, followed by either treatment with 2.5uM veliparib (PARPi) or vehicle control (DMSO) for for 16 hours. Top, right: Immunoblot with the indicated antisera. Bottom: Volcano plots of transcripts found to be differentially regulated PARPi v. vehicle control. Red dots indicate transcripts that were both statistically significantly altered (p<0.05) and more than 1.5-fold changedGenes found to be down-regulated by PARPi as described above (p value < 0.05, 1.5-fold change) in either HT-sensitive cells (left) or CRPC cells (right) were queried against the expression of these genes in the Grasso et al data set in Oncomine. Benign = grey, primary PCa = blue, metastases = orange. Boxplot was generated using the mean expression of the PARPi down-regulated genes in the indicated data sets. Statistical significance determined by two-tailed Student"s t


Figure 3Indicated cell lines were treated as depicted in Figure 2 . Data are depicted as mean + / - standard deviation of three independent biological experiments . Statistical significance was determine by two - tailed Student " s t test where * = p < 0 . 05 , * * = p < 0 . 01 , * * * = p < 0 . 001 , * * * * = p < 0 . 0001 . LNCaP : E2F1 , p = 0 . 0159 ; PCNA , p = 0 . 0217 ; MCM7 , p = 4 . 0936e - 6 ; CCNA2 , p = 0 . 0005 . C4 - 2 : E2F1 , p = 0 . 0074 ; PCNA , p = 0 . 1258 ; MCM7 , p = 3 . 7471e - 5 ; CCNA2 , p = 0ChIP - qPCR after C4 - 2 cells were treated as depicted in Figure 2 . Data are depicted as mean + / - standard deviation of three independent biological experimentsAthymic nude mice were injected with C4 - 2 cell mixed with matrigel . Once tumors became 100mm3 , mice were treated with either vehicle control or veliparib . 72 hours later , tumors were harvested , RNA was isolated and used for qPCR quantification of the indicated transcripts . Data are depicted as log2 absolute gene regulation of veliparib samples compared to control samples , + / - standard deviation of three independent xenograft tumorsProstatectomy tissue ( n = 6 ) was cultured as previously described , and treated with either vehicle control or veliparib for six days . RNA was then harvested from the tissues and used for qPCR quantification of the indicated transcripts . Data are depicted as log2 absolute gene regulation of veliparib samples compared to control samples Each individual tissue is depicted by a separate bar

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Figure 3Indicated cell lines were treated as depicted in Figure 2. Data are depicted as mean +/- standard deviation of three independent biological experiments. Statistical significance was determine by two-tailed Student"s t test where *=p<0.05, **=p<0.01,***=p<0.001, ****=p<0.0001. LNCaP: E2F1, p=0.0159; PCNA, p=0.0217; MCM7, p=4.0936e-6; CCNA2, p=0.0005. C4-2: E2F1, p=0.0074; PCNA, p=0.1258; MCM7, p=3.7471e-5; CCNA2, p=0ChIP-qPCR after C4-2 cells were treated as depicted in Figure 2. Data are depicted as mean +/- standard deviation of three independent biological experimentsAthymic nude mice were injected with C4-2 cell mixed with matrigel. Once tumors became 100mm3, mice were treated with either vehicle control or veliparib. 72 hours later, tumors were harvested, RNA was isolated and used for qPCR quantification of the indicated transcripts. Data are depicted as log2 absolute gene regulation of veliparib samples compared to control samples, +/- standard deviation of three independent xenograft tumorsProstatectomy tissue (n=6) was cultured as previously described, and treated with either vehicle control or veliparib for six days. RNA was then harvested from the tissues and used for qPCR quantification of the indicated transcripts. Data are depicted as log2 absolute gene regulation of veliparib samples compared to control samples Each individual tissue is depicted by a separate bar color


Figure 4Indicated cell lines were treated as depicted in Figure 2 , and labeled with bromodeoxyuridine ( BrdU ) , harvested at indicated time points and utilized for FACS

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Figure 4Indicated cell lines were treated as depicted in Figure 2, and labeled with bromodeoxyuridine (BrdU), harvested at indicated time points and utilized for FACS analyses


Figure 5Left : Data generated as described above in Figure 2 was used to generate a heatmap of homologous recombination ( HR ) gene expression after the indicated treatment regimens . Middle : Selected GSEA MSigDB Oncogenic Signature pathways are shown . Right : Data generated as described above in Figure 2 was compared to a previously described HR deficiency transcriptional profile ( Peng et al . , Nature Communications , 2014 ) . This profile was derived by independently silencing either BRCA1 , RAD51 , or BRIP1 , followed by transcriptional analyses . The union of these three data sets was used to generate the signature . Cut - offs for comparison were a p value < 0 . 05 , and fold change of 1 . 5 . Venn diagrams show the overlapping and non - overlapping genes of both down - ( top ) and up - regulated ( bottom ) genes in the previously - defined HR deficiency signature , and the PARPi - responsive transcriptomeLeft : C4 - 2 cells were treated as depicted in Figure 2 . Data are depicted as mean + / - standard deviation of three independent biological experiments . Statistical significance was determined by two - tailed Student " s t test where * = p < 0 . 05 , * * = p < 0 . 01 . BRCA2 , p = 0 . 0046 ; RAD51 , p = 0 . 0151 ; XRCC3 , p = 0 . 0341 ; TOP3A , p = 0 . 04988 . Right : C4 - 2 cells were treated as depicted in Figure 2 and immunoblotted with the indicated antisera . Quantifications shown below eachAthymic nude mice were injected with C4 - 2 cell mixed with matrigel . Once tumors became 100mm3 , mice were treated with either vehicle control or veliparib . 72 hours later , tumors were harvested , RNA was isolated and used for qPCR quantification of the indicated transcripts . Data are depicted as log2 absolute gene regulation of veliparib samples compared to control samples , + / - standard deviation of three independent xenograft tumorsProstatectomy tissue ( n = 6 ) was cultured as previously described , and treated with either vehicle control or veliparib for six days . RNA was then harvested from the tissues and used for qPCR quantification of the indicated transcripts . Data are depicted as log2 absolute gene regulation of veliparib samples compared to control samples . Each individual tissue is depicted by a separate bar color . Statistical analyses were performed by Wilcoxon signed rank testC4 - 2 cells were treated as depicted in Figure 2 . ChIP was performed using the indicated antisera , and the subsequent DNA was isolated and used in qPCR reaction using primers designed to amplify the indicated genomic loci : BRCA2 enhancer , RAD51 promoter , or TOP3A promoterC4 - 2 cells treated with 2 . 5uM veliparib ( Vel . ) or vehicle control ( Veh . ) for 24 hours . Cells were then harvested , lysed , and differentially centrifuged as described in the material and methods section , resulting in a soluble fraction ( Sol . ) ( GAPDH serves as control ) or a chromatin - tethered fraction ( Teth . ) ( histone H4 serves as control ) . Immunoblots were performed for the indicated

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Figure 5Left: Data generated as described above in Figure 2 was used to generate a heatmap of homologous recombination (HR) gene expression after the indicated treatment regimens. Middle: Selected GSEA MSigDB Oncogenic Signature pathways are shown. Right: Data generated as described above in Figure 2 was compared to a previously described HR deficiency transcriptional profile (Peng et al., Nature Communications, 2014). This profile was derived by independently silencing either BRCA1, RAD51, or BRIP1, followed by transcriptional analyses. The union of these three data sets was used to generate the signature. Cut-offs for comparison were a p value<0.05, and fold change of 1.5. Venn diagrams show the overlapping and non-overlapping genes of both down- (top) and up-regulated (bottom) genes in the previously-defined HR deficiency signature, and the PARPi-responsive transcriptomeLeft: C4-2 cells were treated as depicted in Figure 2. Data are depicted as mean +/- standard deviation of three independent biological experiments. Statistical significance was determined by two-tailed Student"s t test where *=p<0.05, **=p<0.01. BRCA2, p=0.0046; RAD51, p=0.0151; XRCC3, p=0.0341; TOP3A, p=0.04988. Right: C4-2 cells were treated as depicted in Figure 2 and immunoblotted with the indicated antisera. Quantifications shown below eachAthymic nude mice were injected with C4-2 cell mixed with matrigel. Once tumors became 100mm3, mice were treated with either vehicle control or veliparib. 72 hours later, tumors were harvested, RNA was isolated and used for qPCR quantification of the indicated transcripts. Data are depicted as log2 absolute gene regulation of veliparib samples compared to control samples, +/- standard deviation of three independent xenograft tumorsProstatectomy tissue (n=6) was cultured as previously described, and treated with either vehicle control or veliparib for six days. RNA was then harvested from the tissues and used for qPCR quantification of the indicated transcripts. Data are depicted as log2 absolute gene regulation of veliparib samples compared to control samples. Each individual tissue is depicted by a separate bar color. Statistical analyses were performed by Wilcoxon signed rank testC4-2 cells were treated as depicted in Figure 2. ChIP was performed using the indicated antisera, and the subsequent DNA was isolated and used in qPCR reaction using primers designed to amplify the indicated genomic loci: BRCA2 enhancer, RAD51 promoter, or TOP3A promoterC4-2 cells treated with 2.5uM veliparib (Vel.) or vehicle control (Veh.) for 24 hours. Cells were then harvested, lysed, and differentially centrifuged as described in the material and methods section, resulting in a soluble fraction (Sol.) (GAPDH serves as control) or a chromatin-tethered fraction (Teth.) (histone H4 serves as control). Immunoblots were performed for the indicated proteins


Figure 6Expression levels of indicated HR pathway genes in primary PCa vs normal patient samples . Violin plots represent FPKM normalized counts obtained from matched tumor and normal RNA - Seq data from TCGA ( n = 52 ) with p - values generated using paired t -

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Figure 6Expression levels of indicated HR pathway genes in primary PCa vs normal patient samples. Violin plots represent FPKM normalized counts obtained from matched tumor and normal RNA-Seq data from TCGA (n=52) with p-values generated using paired t-tests


Figure 7Indicated cell lines were transfected with indicated constructs , and treated with veliparib . Cell growt was assessed LNCaP cell growth : Control transfection , p = 0 . 0220 ; BRCA1 transfection , p = 0 . 67787 ; BRCA2 transfection , p = 0 . 4676 . C4 - 2 cell growth : Control transfection , p = 0 . 0354 ; BRCA1 transfection , p = 0 . 1638 ; BRCA2 transfection , p = 0 . 2519 . 22Rv1 cell growth : Control transfection , p = 0 . 0039 ; BRCA1 transfection , p = 0 . 1085 ; BRCA2 transfection , p = 0 . 2781Indicated cell lines were transfected with indicated constructs , and treated with veliparib DDR via γH2AX was assessed LNCaP γH2AX : Control transfection , p = 0 . 0008 ; BRCA1 transfection , p = 0 . 9035 ; BRCA2 transfection , p = 0 . 4685 . C4 - 2 γH2AX : Control transfection , p = 0 . 0009 ; BRCA1 transfection , p = 0 . 6362 ; BRCA2 transfection , p = 0 . 4217 . 22Rv1 γH2AX : Control transfection , p < 0 . 0001 ; BRCA1 transfection , p = 0 . 4698 ; BRCA2 transfection , p = 0 .

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Figure 7Indicated cell lines were transfected with indicated constructs, and treated with veliparib. Cell growt was assessed LNCaP cell growth: Control transfection, p=0.0220; BRCA1 transfection, p=0.67787; BRCA2 transfection, p=0.4676. C4-2 cell growth: Control transfection, p=0.0354; BRCA1 transfection, p=0.1638; BRCA2 transfection, p=0.2519. 22Rv1 cell growth: Control transfection, p=0.0039; BRCA1 transfection, p=0.1085; BRCA2 transfection, p=0.2781Indicated cell lines were transfected with indicated constructs, and treated with veliparib DDR via γH2AX was assessed LNCaP γH2AX: Control transfection, p=0.0008; BRCA1 transfection, p=0.9035; BRCA2 transfection, p=0.4685. C4-2 γH2AX: Control transfection, p=0.0009; BRCA1 transfection, p=0.6362; BRCA2 transfection, p=0.4217. 22Rv1 γH2AX: Control transfection, p<0.0001; BRCA1 transfection, p=0.4698; BRCA2 transfection, p=0.4937


Figure 1 ( B ) DNA knotting probability ( Pkn ) of intracellular chromatin ( observed ) does not scale proportionally to the length of DNA ( expected ) . The slope of Pkn is reduced in chromatin stretches larger than 20 nucleosomes . Data from ( Valdes et al . , 2018 ) .

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Figure 1(B) DNA knotting probability (Pkn) of intracellular chromatin (observed) does not scale proportionally to the length of DNA (expected). The slope of Pkn is reduced in chromatin stretches larger than 20 nucleosomes. Data from (Valdes et al., 2018).


Figure 2 ( B ) Lanes 1 and 2 : negatively supercoiled plasmid ( YEp24 ) and its equilibrium distribution of Lk topoisomers upon its relaxation with Topo I . Lanes 3 and 4 : distribution of Lk topoisomers of YEp24 upon its relaxation in lysates of ∆ top1 TOP2 yeast cells in absence and after the addition of ICRF - 193 . Plots compare the relative intensity of individual topoisomers of the Lk distributions in lanes 2 , 3 and 4 . ( C ) Top : 2D gel electrophoresis of the Lk distributions of the YEp13 minichromosome present in the lysates of ∆ top1 TOP2 yeast cells before and after the addition of ICRF - 193 ( see details in Fig EV3 ) . Plots compare the relative intensity of the Lk distributions ( divided into ten sections ) . Bottom : 2D gel electrophoresis of the same samples upon nicking the DNA in order to reveal the occurrence of knots ( see details in Fig EV4 ) . The graph shows Pkn of YEp13 ( mean ± SD from three independent experiments ) . P - values ( Student ' s t test ) : ns , p > 0 . 05 . ( E ) Lk distributions of the control plasmid ( YEp24 ) relaxed by lysates of ∆ top1 top2 - 4 yeast cells that expressed TOP2 or Top2 - ∆ 83 . Plots compare the relative intensity of individual Lk topoisomers . ( F ) Top : 2D gel electrophoresis of the Lk distributions of the YEp13 minichromosome produced in the presence of TOP2 or Top2 - ∆ 83 . Plots compare the relative intensity of the Lk distributions ( divided into ten sections ) . Bottom : 2D gel electrophoresis of the same samples upon nicking the DNA in order to reveal the occurrence of knots . Graph : Pkn of YEp13 ( mean ± SD from three independent experiments ) .

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Figure 2(B) Lanes 1 and 2: negatively supercoiled plasmid (YEp24) and its equilibrium distribution of Lk topoisomers upon its relaxation with Topo I. Lanes 3 and 4: distribution of Lk topoisomers of YEp24 upon its relaxation in lysates of ∆top1 TOP2 yeast cells in absence and after the addition of ICRF-193. Plots compare the relative intensity of individual topoisomers of the Lk distributions in lanes 2, 3 and 4.(C) Top: 2D gel electrophoresis of the Lk distributions of the YEp13 minichromosome present in the lysates of ∆top1 TOP2 yeast cells before and after the addition of ICRF-193 (see details in Fig EV3). Plots compare the relative intensity of the Lk distributions (divided into ten sections). Bottom: 2D gel electrophoresis of the same samples upon nicking the DNA in order to reveal the occurrence of knots (see details in Fig EV4). The graph shows Pkn of YEp13 (mean ± SD from three independent experiments). P-values (Student's t test): ns, p > 0.05.(E) Lk distributions of the control plasmid (YEp24) relaxed by lysates of ∆top1 top2-4 yeast cells that expressed TOP2 or Top2-∆83. Plots compare the relative intensity of individual Lk topoisomers.(F) Top: 2D gel electrophoresis of the Lk distributions of the YEp13 minichromosome produced in the presence of TOP2 or Top2-∆83. Plots compare the relative intensity of the Lk distributions (divided into ten sections). Bottom: 2D gel electrophoresis of the same samples upon nicking the DNA in order to reveal the occurrence of knots. Graph: Pkn of YEp13 (mean ± SD from three independent experiments).


Figure 3 ( A ) Top , DNA content ( n / 2n ) of exponentially growing ( OD600 = 0 . 6 - 0 . 8 ) smc2 - 8 yeast cells . First blot : 2D gel electrophoresis of the distribution of Lk topoisomers ( Lk ) of the YEp13 DNA in cells quenched at 26oC and after shifting the culture to 35oC for 60 min . Second blot : 2D gel electrophoresis of the same samples upon nicking the YEp13 DNA in order to reveal the occurrence of knots ( kn ) . Graph : Pkn of YEp13 before and after the inactivation of condensin . Top , DNA content ( n / 2n ) of exponentially growing yeast cells . First blot : 2D gel electrophoresis of the distribution of Lk topoisomers ( Lk ) of the YEp13 DNA in cells quenched at 26oC and after shifting the culture to 35oC for 60 min . Second blot : 2D gel electrophoresis of the same samples upon nicking the YEp13 DNA in order to reveal the occurrence of knots ( kn ) . ( B ) Experiments conducted as in ( A ) but in scc1 - 73 yeast cells . Graph : Pkn of YEp13 before and after the inactivation of cohesin . Top , DNA content ( n / 2n ) of exponentially growing yeast cells . First blot : 2D gel electrophoresis of the distribution of Lk topoisomers ( Lk ) of the YEp13 DNA in cells quenched at 26oC and after shifting the culture to 35oC for 60 min . Second blot : 2D gel electrophoresis of the same samples upon nicking the YEp13 DNA in order to reveal the occurrence of knots ( kn ) . ( C ) Experiments conducted as in ( A ) but in smc6 - 9 yeast cells . Graph : Pkn of YEp13 before and after the inactivation of the Smc5 / 6 complex . ( D , E ) Experiments conducted as in ( A ) , but in cells arrested in G1 with alfa - factor ( D ) or in metaphase with nocodazole ( E ) for 2 h at 26oC and for one additional hour at 26oC or 35oC . ( F , G ) Experiments conducted as in ( B ) , but in cells arrested in G1 with alfa - factor for 2 h at 26oC ( F ) or in metaphase with nocodazole ( G ) and for one additional hour at 26oC or 35oC . ( H , I ) Experiments conducted as in ( C ) , but in cells arrested in G1 with alfa - factor ( H ) or in metaphase with nocodazole ( I ) for 2 h at 26oC and for one additional hour at 26oC or 35oC .

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Figure 3(A) Top, DNA content (n/2n) of exponentially growing (OD600 = 0.6-0.8) smc2-8 yeast cells. First blot: 2D gel electrophoresis of the distribution of Lk topoisomers (Lk) of the YEp13 DNA in cells quenched at 26oC and after shifting the culture to 35oC for 60 min. Second blot: 2D gel electrophoresis of the same samples upon nicking the YEp13 DNA in order to reveal the occurrence of knots (kn). Graph: Pkn of YEp13 before and after the inactivation of condensin.Top, DNA content (n/2n) of exponentially growing yeast cells. First blot: 2D gel electrophoresis of the distribution of Lk topoisomers (Lk) of the YEp13 DNA in cells quenched at 26oC and after shifting the culture to 35oC for 60 min. Second blot: 2D gel electrophoresis of the same samples upon nicking the YEp13 DNA in order to reveal the occurrence of knots (kn). (B) Experiments conducted as in (A) but in scc1-73 yeast cells. Graph: Pkn of YEp13 before and after the inactivation of cohesin.Top, DNA content (n/2n) of exponentially growing yeast cells. First blot: 2D gel electrophoresis of the distribution of Lk topoisomers (Lk) of the YEp13 DNA in cells quenched at 26oC and after shifting the culture to 35oC for 60 min. Second blot: 2D gel electrophoresis of the same samples upon nicking the YEp13 DNA in order to reveal the occurrence of knots (kn). (C) Experiments conducted as in (A) but in smc6-9 yeast cells. Graph: Pkn of YEp13 before and after the inactivation of the Smc5/6 complex.(D, E) Experiments conducted as in (A), but in cells arrested in G1 with alfa-factor (D) or in metaphase with nocodazole (E) for 2 h at 26oC and for one additional hour at 26oC or 35oC.(F, G) Experiments conducted as in (B), but in cells arrested in G1 with alfa-factor for 2 h at 26oC (F) or in metaphase with nocodazole (G) and for one additional hour at 26oC or 35oC.(H, I) Experiments conducted as in (C), but in cells arrested in G1 with alfa-factor (H) or in metaphase with nocodazole (I) for 2 h at 26oC and for one additional hour at 26oC or 35oC.


Figure 4 ( A - E ) DNA topology of the indicated minichromosomes of increasing DNA length ( kb ) before ( 26oC ) and after inactivation of condensin ( 35oC ) in smc2 - 8 cells . In each case , the first 2D gel resolves the Lk topoisomers ( Lk ) , the second 2D gel uncovers the knotted forms ( Kn ) . Gel signals are denoted as in Fig 2 . Bottom graphs compare the Pkn before and after the inactivation of condensin ( mean ± SD from three independent experiments ) . P - values ( Student ' s t test ) : ns , p > 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . ( F ) Plot of Pkn values of minichromosomes of increasing DNA length ( including YEp13 ) before and after inactivation of condensin .

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

Figure 4(A-E) DNA topology of the indicated minichromosomes of increasing DNA length (kb) before (26oC) and after inactivation of condensin (35oC) in smc2-8 cells. In each case, the first 2D gel resolves the Lk topoisomers (Lk), the second 2D gel uncovers the knotted forms (Kn). Gel signals are denoted as in Fig 2. Bottom graphs compare the Pkn before and after the inactivation of condensin (mean ± SD from three independent experiments). P-values (Student's t test): ns, p > 0.05; **p < 0.01; ***p < 0.001. (F) Plot of Pkn values of minichromosomes of increasing DNA length (including YEp13) before and after inactivation of condensin.


Figure 5 ( A - D ) DNA topology of the indicated minichromosomes of increasing DNA length ( kb ) before ( 26oC ) and after inactivation of cohesin ( 35oC ) in scc1 - 73 cells . In each case , the first 2D gel resolves the Lk topoisomers ( Lk ) , the second 2D gel uncovers the knotted forms ( Kn ) . Gel signals are denoted as in Fig 2 . Bottom graphs compare the Pkn before and after the inactivation of cohesin ( mean ± SD from three independent experiments ) . P - values ( Student ' s t test ) : ns , p > 0 . 05 . ( E ) Plot of Pkn values of minichromosomes of increasing DNA length ( including YEp13 ) before and after inactivation of cohesin . P - values ( Student ' s t test ) : * * p < 0 . 01 .

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

Figure 5(A-D) DNA topology of the indicated minichromosomes of increasing DNA length (kb) before (26oC) and after inactivation of cohesin (35oC) in scc1-73 cells. In each case, the first 2D gel resolves the Lk topoisomers (Lk), the second 2D gel uncovers the knotted forms (Kn). Gel signals are denoted as in Fig 2. Bottom graphs compare the Pkn before and after the inactivation of cohesin (mean ± SD from three independent experiments). P-values (Student's t test): ns, p > 0.05.(E) Plot of Pkn values of minichromosomes of increasing DNA length (including YEp13) before and after inactivation of cohesin. P-values (Student's t test): **p < 0.01.


Figure 6 ( A - D ) DNA topology of the indicated minichromosomes of increasing DNA length ( kb ) before ( 26oC ) and after inactivation of Smc5 / 6 complex ( 35oC ) in smc6 - 9 cells . In each case , the first 2D gel resolves the Lk topoisomers ( Lk ) , the second 2D gel uncovers the knotted forms ( Kn ) . Gel signals are denoted as in Fig 2 . Bottom graphs compare the Pkn before and after the inactivation of Smc5 / 6 complex ( mean ± SD from two independent experiments ) . ( E ) Plot of Pkn values of minichromosomes of increasing DNA length ( including YEp13 ) before and after inactivation of Smc5 / 6 complex .

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

Figure 6(A-D) DNA topology of the indicated minichromosomes of increasing DNA length (kb) before (26oC) and after inactivation of Smc5/6 complex (35oC) in smc6-9 cells. In each case, the first 2D gel resolves the Lk topoisomers (Lk), the second 2D gel uncovers the knotted forms (Kn). Gel signals are denoted as in Fig 2. Bottom graphs compare the Pkn before and after the inactivation of Smc5/6 complex (mean ± SD from two independent experiments).(E) Plot of Pkn values of minichromosomes of increasing DNA length (including YEp13) before and after inactivation of Smc5/6 complex.


Figure 1A Measurement of urinary albumin / creatinine by ELISA ( albumin - to - creatinine ratio : day 14 : Phb2fl / fl 0 . 09 ± 0 . 03 mg / mg , n = 4 , versus Phb2pko 0 . 37 ± 0 . 21 mg / mg , n = 4 , P = 0 . 2350 ; day 21 : Phb2fl / fl 0 . 65 ± 0 . 16 mg / mg , n = 4 , versus Phb2pko 193 . 10 ± 26 . 81 mg / mg , n = 4 , * * * P = 0 . 003 ; day 28 : Phb2fl / fl 0 . 57 ± 0 . 07 mg / mg , n = 4 , versus Phb2pko 470 . 40 ± 131 . 30 mg / mg , n = 4 , * P = 0 . 0117 ) . B Appearance of Phb2fl / fl and Phb2pko mice at day 28 . C Analysis of body weight from postnatal day 14 until day 32 ( P - values for Phb2fl / fl or Phb2het versus Phb2pko : * P = 0 . 0372 at day 27 , * P = 0 . 0311 at day 28 , * P = 0 . 0157 at day 29 , * * P = 0 . 008 at day 30 , * * P = 0 . 0013 at day 31 , * * P = 0 . 0035 at day 32 , n = 3 for Phb2fl / fl and n = 4 for Phb2het and Phb2pko ) . D Measurement of serum creatinine levels of mice in their fifth week of life ( Phb2fl / fl / het 14 . 74 ± 3 . 75 μmol / l versus Phb2pko 74 . 73 ± 2 . 20 μmol / l , n = 3 for both groups ; * * * P = 0 . 0002 ) . E Measurement of serum urea levels of mice in their fifth week of life ( Phb2fl / fl / het 44 . 00 ± 7 . 02 mg / dl versus Phb2pko 493 . 70 ± 45 . 34 mg / dl , n = 3 for both groups ; * * * P = 0 . 006 ) . F Kaplan - Meier survival curve ( n = 5 for Phb2fl / fl / het and n = 6 for Phb2pko ) .

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

Figure 1A Measurement of urinary albumin/creatinine by ELISA (albumin-to-creatinine ratio: day 14: Phb2fl/fl 0.09 ± 0.03 mg/mg, n = 4, versus Phb2pko 0.37 ± 0.21 mg/mg, n = 4, P = 0.2350; day 21: Phb2fl/fl 0.65 ± 0.16 mg/mg, n = 4, versus Phb2pko 193.10 ± 26.81 mg/mg, n = 4, ***P = 0.003; day 28: Phb2fl/fl 0.57 ± 0.07 mg/mg, n = 4, versus Phb2pko 470.40 ± 131.30 mg/mg, n = 4, *P = 0.0117).B Appearance of Phb2fl/fl and Phb2pko mice at day 28.C Analysis of body weight from postnatal day 14 until day 32 (P-values for Phb2fl/fl or Phb2het versus Phb2pko: *P = 0.0372 at day 27, *P = 0.0311 at day 28, *P = 0.0157 at day 29, **P = 0.008 at day 30, **P = 0.0013 at day 31, **P = 0.0035 at day 32, n = 3 for Phb2fl/fl and n = 4 for Phb2het and Phb2pko).D Measurement of serum creatinine levels of mice in their fifth week of life (Phb2fl/fl/het 14.74 ± 3.75 μmol/l versus Phb2pko 74.73 ± 2.20 μmol/l, n = 3 for both groups; ***P = 0.0002).E Measurement of serum urea levels of mice in their fifth week of life (Phb2fl/fl/het 44.00 ± 7.02 mg/dl versus Phb2pko 493.70 ± 45.34 mg/dl, n = 3 for both groups; ***P = 0.006).F Kaplan-Meier survival curve (n = 5 for Phb2fl/fl/het and n = 6 for Phb2pko).


Figure 2Phb2pko mice develop glomerulosclerosisA PAS staining of kidney sections ( scale bar : 20 μm ) . B Immunohistochemistry for podocin on kidney sections ( scale bar : 20 μm ) . C Analysis of podocyte foot processes in electron micrographs ( scale bar : 0 . 7 μm ) . FP , podocyte foot processes ; GBM , glomerular basement membrane ; EC , endothelial cells . D Analysis of mitochondrial morphology in electron micrographs ( arrows ; scale bar : 0 . 3 μm ) . E High - power view of mitochondria in electron micrographs ( scale bar : 0 . 1 μm ) .

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Figure 2Phb2pko mice develop glomerulosclerosisA PAS staining of kidney sections (scale bar: 20 μm).B Immunohistochemistry for podocin on kidney sections (scale bar: 20 μm).C Analysis of podocyte foot processes in electron micrographs (scale bar: 0.7 μm). FP, podocyte foot processes; GBM, glomerular basement membrane; EC, endothelial cells.D Analysis of mitochondrial morphology in electron micrographs (arrows; scale bar: 0.3 μm).E High-power view of mitochondria in electron micrographs (scale bar: 0.1 μm).


Figure 3A PAS staining of tPod - Phb2pko and control mice 2 . 5 weeks after the end of tamoxifen treatment ( scale bar : 20 μm ) . B Immunohistochemistry for podocin on kidney sections of tPod - Phb2pko and control mice 2 . 5 weeks after the end of tamoxifen treatment ( scale bar : 20 μm ) . C Coomassie stain of urinary samples of tPod - Phb2pko and control mice .

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Figure 3A PAS staining of tPod-Phb2pko and control mice 2.5 weeks after the end of tamoxifen treatment (scale bar: 20 μm).B Immunohistochemistry for podocin on kidney sections of tPod-Phb2pko and control mice 2.5 weeks after the end of tamoxifen treatment (scale bar: 20 μm).C Coomassie stain of urinary samples of tPod-Phb2pko and control mice.


Figure 4A Kaplan - Meier survival curve showed that an additional knockout of the insulin receptor ( Insr ) prolonged survival of Phb2pko mice ( n = 7 for Phb2pko / Insrpko , n = 4 for Phb2pko / Insrhet , and n = 6 for Phb2pko ) . B Kaplan - Meier survival curve revealed that the survival time of Phb2pko mice is not changed by an additional Igf1r deficiency ( n = 6 for Phb2pko / Igf1rpko , n = 4 for Phb2pko / Igf1rhet , and n = 6 for Phb2pko ) . C Statistical analysis comparing all genotypes with Phb2pko mice . D Kaplan - Meier survival curve revealed prolonged survival of Phb2pko / Insrpko / Igf1rpko and Phb2pko / Insrpko / Igf1rhet mice ( n = 19 for Phb2pko / Insrpko / Igf1rpko , n = 9 for Phb2pko / Insrhet / Igf1rpko , n = 11 for Phb2pko / Insrpko / Igf1rhet , n = 9 for Phb2pko / Insrhet / Igf1rhet , and n = 6 for Phb2pko ) . E Statistical analysis comparing all genotypes with Phb2pko mice .

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

Figure 4A Kaplan-Meier survival curve showed that an additional knockout of the insulin receptor (Insr) prolonged survival of Phb2pko mice (n = 7 for Phb2pko/Insrpko, n = 4 for Phb2pko/Insrhet, and n = 6 for Phb2pko).B Kaplan-Meier survival curve revealed that the survival time of Phb2pko mice is not changed by an additional Igf1r deficiency (n = 6 for Phb2pko/Igf1rpko, n = 4 for Phb2pko/Igf1rhet, and n = 6 for Phb2pko).C Statistical analysis comparing all genotypes with Phb2pko mice.D Kaplan-Meier survival curve revealed prolonged survival of Phb2pko/Insrpko/Igf1rpko and Phb2pko/Insrpko/Igf1rhet mice (n = 19 for Phb2pko/Insrpko/Igf1rpko, n = 9 for Phb2pko/Insrhet/Igf1rpko, n = 11 for Phb2pko/Insrpko/Igf1rhet, n = 9 for Phb2pko/Insrhet/Igf1rhet, and n = 6 for Phb2pko).E Statistical analysis comparing all genotypes with Phb2pko mice.


Figure 5A Serum creatinine and urea levels in the fifth week of life ( left graph : Phb2pko 74 . 73 ± 2 . 20 μmol / l versus Phb2pko / Insrpko / Igf1rpko 37 . 36 ± 8 . 83 μmol / l , n = 3 for Phb2pko and n = 4 for Phb2pko / Insrpko / Igf1rpko , * P = 0 . 0169 ; right graph : Phb2pko 493 . 70 ± 45 . 34 mg / dl versus Phb2pko / Insrpko / Igf1rpko 272 . 80 ± 49 . 29 mg / dl , n = 3 for Phb2pko and n = 4 for Phb2pko / Insrpko / Igf1rpko , * P = 0 . 0247 ) . B Abundance of deleted Phb2 gene in isolated glomeruli of Phb2fl / fl , Phb2het , Phb2pko and Phb2pko / Insrpko / Igf1rpko mice compared to the floxed Phb2 gene ( left graph ; quantified by qPCR : n = 3 for all groups , P = 0 . 83 for Phb2pko / Insrpko / Igf1rpko versus Phb2pko ) and compared to a reference gene ( right graph ; quantified by qPCR : n = 3 for all groups , P = 0 . 58 for Phb2pko / Insrpko / Igf1rpko versus Phb2pko ) . C Analysis of mitochondrial morphology in electron micrographs ( left image : scale bar , 1 μm ; right image : scale bar , 0 . 1 μm ) .

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

Figure 5A Serum creatinine and urea levels in the fifth week of life (left graph: Phb2pko 74.73 ± 2.20 μmol/l versus Phb2pko/Insrpko/Igf1rpko 37.36 ± 8.83 μmol/l, n = 3 for Phb2pko and n = 4 for Phb2pko/Insrpko/Igf1rpko, *P = 0.0169; right graph: Phb2pko 493.70 ± 45.34 mg/dl versus Phb2pko/Insrpko/Igf1rpko 272.80 ± 49.29 mg/dl, n = 3 for Phb2pko and n = 4 for Phb2pko/Insrpko/Igf1rpko, *P = 0.0247).B Abundance of deleted Phb2 gene in isolated glomeruli of Phb2fl/fl, Phb2het, Phb2pko and Phb2pko/Insrpko/Igf1rpko mice compared to the floxed Phb2 gene (left graph; quantified by qPCR: n = 3 for all groups, P = 0.83 for Phb2pko/Insrpko/Igf1rpko versus Phb2pko) and compared to a reference gene (right graph; quantified by qPCR: n = 3 for all groups, P = 0.58 for Phb2pko/Insrpko/Igf1rpko versus Phb2pko).C Analysis of mitochondrial morphology in electron micrographs (left image: scale bar, 1 μm; right image: scale bar, 0.1 μm).


Figure 6A Immunofluorescence staining of mPhb2 shRNA and control podocytes with a Tom20 antibody revealed a disturbed mitochondrial network in Phb2 - deficient podocytes ( scale bar : 10 μm ) . B Quantification of tubular and fragmented mitochondrial morphology mPhb2 shRNA versus control podocytes seen in ( A ) ( mitochondrial network of n = 100 individual cells of mPhb2 shRNA podocytes and n = 108 individual cells of scrambled shRNA podocytes was assessed ) . C Morphometric image analysis revealed an increased ratio of mitochondrial count versus mitochondrial area in Phb2 - deficient podocytes compared to control podocytes ( n = 75 cell patches for scrambled shRNA , n = 58 cell patches for mPhb2 shRNA ) . D Morphometric image analysis showed a decrease in branch numbers in Phb2 - deficient podocytes compared to control podocytes ( n = 75 cell patches for scrambled shRNA , n = 58 cell patches for mPhb2 shRNA ) . E Morphometric image analysis detected a decrease in mitochondrial size in Phb2 - deficient podocytes compared to control podocytes ( n = 75 cell patches for scrambled shRNA , n = 58 cell patches for mPhb2 shRNA ) . F Measurements of mitochondrial respiration with complex II substrates ( succinate and glycerol - 3 - phosphate ) did not show significant differences between Phb2 - deficient and control podocytes ( n = 6 ) . G Quantification of mtDNA as a measure for mitochondrial mass showed similar levels of mtDNA in Phb2 - deficient podocytes compared to control podocytes ( n = 3 ) . H FACS analysis of MitoTracker - and MitoSOX - stained Phb2 - deficient podocytes and control podocytes . I Quantification of mean fluorescence intensity of MitoTracker and MitoSOX by FACS analysis revealed the same mitochondrial mass and the same ROS levels in Phb2 - deficient podocytes compared to control podocytes ( n = 3 , P = 0 . 7782 for MitoTracker , P = 0 . 1088 for MitoSOX ) .

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

Figure 6A Immunofluorescence staining of mPhb2 shRNA and control podocytes with a Tom20 antibody revealed a disturbed mitochondrial network in Phb2-deficient podocytes (scale bar: 10 μm).B Quantification of tubular and fragmented mitochondrial morphology mPhb2 shRNA versus control podocytes seen in (A) (mitochondrial network of n = 100 individual cells of mPhb2 shRNA podocytes and n = 108 individual cells of scrambled shRNA podocytes was assessed).C Morphometric image analysis revealed an increased ratio of mitochondrial count versus mitochondrial area in Phb2-deficient podocytes compared to control podocytes (n = 75 cell patches for scrambled shRNA, n = 58 cell patches for mPhb2 shRNA).D Morphometric image analysis showed a decrease in branch numbers in Phb2-deficient podocytes compared to control podocytes (n = 75 cell patches for scrambled shRNA, n = 58 cell patches for mPhb2 shRNA).E Morphometric image analysis detected a decrease in mitochondrial size in Phb2-deficient podocytes compared to control podocytes (n = 75 cell patches for scrambled shRNA, n = 58 cell patches for mPhb2 shRNA).F Measurements of mitochondrial respiration with complex II substrates (succinate and glycerol-3-phosphate) did not show significant differences between Phb2-deficient and control podocytes (n = 6).G Quantification of mtDNA as a measure for mitochondrial mass showed similar levels of mtDNA in Phb2-deficient podocytes compared to control podocytes (n = 3).H FACS analysis of MitoTracker- and MitoSOX-stained Phb2-deficient podocytes and control podocytes.I Quantification of mean fluorescence intensity of MitoTracker and MitoSOX by FACS analysis revealed the same mitochondrial mass and the same ROS levels in Phb2-deficient podocytes compared to control podocytes (n = 3, P = 0.7782 for MitoTracker, P = 0.1088 for MitoSOX).


Figure 7A Phb2 - deficient mouse podocytes showed higher levels of phosphorylated S6 ribosomal protein than control podocytes , which could be blocked by treatment with rapamycin ( n = 5 , bars represent mean ± SEM , Student ' s t - test , * * P = 0 . 0036 ) . pS6RP , phosphorylated S6 ribosomal protein ; S6RP , S6 ribosomal protein . B Immunohistochemistry for phosphorylated S6 ribosomal protein ( pS6RP ) on kidney sections revealed increased levels of pS6RP in glomeruli of Phb2pko compared to Phb2fl / fl mice ( arrows point to cells showing a signal for pS6RP ; scale bar : 20 μm ) . C Kaplan - Meier survival curve revealed that treatment of Phb2pko mice with rapamycin prolonged survival for several days compared to vehicle - treated Phb2pko mice ( n = 11 for Phb2fl / fl + rapamycin , n = 15 for Phb2pko + vehicle , n = 16 for Phb2pko + rapamycin ) . D Statistical analysis comparing rapamycin - treated Phb2pko mice with vehicle - treated Phb2pko mice .

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

Figure 7A Phb2-deficient mouse podocytes showed higher levels of phosphorylated S6 ribosomal protein than control podocytes, which could be blocked by treatment with rapamycin (n = 5, bars represent mean ± SEM, Student's t-test, **P = 0.0036). pS6RP, phosphorylated S6 ribosomal protein; S6RP, S6 ribosomal protein.B Immunohistochemistry for phosphorylated S6 ribosomal protein (pS6RP) on kidney sections revealed increased levels of pS6RP in glomeruli of Phb2pko compared to Phb2fl/fl mice (arrows point to cells showing a signal for pS6RP; scale bar: 20 μm).C Kaplan-Meier survival curve revealed that treatment of Phb2pko mice with rapamycin prolonged survival for several days compared to vehicle-treated Phb2pko mice (n = 11 for Phb2fl/fl + rapamycin, n = 15 for Phb2pko + vehicle, n = 16 for Phb2pko + rapamycin).D Statistical analysis comparing rapamycin-treated Phb2pko mice with vehicle-treated Phb2pko mice.


Figure 1D - G Culture dynamics of phage ‐ infected wild ‐ type ( black ) versus BREX ‐ containing ( red ) strains of B . subtilis BEST7003 . Bacterial strains were exposed to phage at time = 0 h , and optical density measurements were read in a 96 ‐ well plate format . Each experiment was performed three times with three technical triplicates for each biological replicate . Error bars represent SEM . Shown are representative results for three of the ten phages tested ; data for the remaining seven phages are found in Supplementary Fig S2 .

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Figure 1D-G Culture dynamics of phage‐infected wild‐type (black) versus BREX‐containing (red) strains of B. subtilis BEST7003. Bacterial strains were exposed to phage at time = 0 h, and optical density measurements were read in a 96‐well plate format. Each experiment was performed three times with three technical triplicates for each biological replicate. Error bars represent SEM. Shown are representative results for three of the ten phages tested; data for the remaining seven phages are found in Supplementary Fig S2.


Figure 2A Culture dynamics over an extended period ( > 30 h ) for SPO1 ‐ infected wild ‐ type ( black ) versus BREX ‐ containing ( red ) strains of B . subtilis BEST7003 . Each curve represents a single technical replicate grown in a single well on a 96 ‐ well plate . Culture decline is temporally reproducible for the BREX ‐ lacking strain but occurs later , at apparently stochastic time points , for the BREX ‐ containing strain . Re ‐ growth following culture crash represents phage ‐ resistant mutants . B Phage production during a one ‐ step phage growth curve experiment with wild ‐ type ( black ) and BREX ‐ containing ( red ) strains of B . subtilis BEST7003 infected with SPO1 . Error bars represent SD . Y ‐ axis represents absolute phage concentrations . Black and red arrows point to the time point of maximal burst for BREX ‐ lacking and BREX ‐ containing strains , respectively . C Phage production during a one ‐ step phage growth curve experiment with wild ‐ type ( black ) and BREX ‐ containing ( red ) strains of B . subtilis BEST7003 infected with Φ3T . Error bars represent SD . Y ‐ axis represents relative phage concentrations normalized to the value at the beginning of the infection . D Multiplex PCR assay showing lysogeny during a phage infection time course in the strain lacking BREX ( black ) , but not in BREX ‐ containing strain ( red ) , or uninfected ( U ) strains . Amplicons for the bacterial DNA , phage DNA , and lysogen ‐ specific DNA are 293 , 485 , and 1 , 218 bp , respectively .

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

Figure 2A Culture dynamics over an extended period (> 30 h) for SPO1‐infected wild‐type (black) versus BREX‐containing (red) strains of B. subtilis BEST7003. Each curve represents a single technical replicate grown in a single well on a 96‐well plate. Culture decline is temporally reproducible for the BREX‐lacking strain but occurs later, at apparently stochastic time points, for the BREX‐containing strain. Re‐growth following culture crash represents phage‐resistant mutants.B Phage production during a one‐step phage growth curve experiment with wild‐type (black) and BREX‐containing (red) strains of B. subtilis BEST7003 infected with SPO1. Error bars represent SD. Y‐axis represents absolute phage concentrations. Black and red arrows point to the time point of maximal burst for BREX‐lacking and BREX‐containing strains, respectively.C Phage production during a one‐step phage growth curve experiment with wild‐type (black) and BREX‐containing (red) strains of B. subtilis BEST7003 infected with Φ3T. Error bars represent SD. Y‐axis represents relative phage concentrations normalized to the value at the beginning of the infection.D Multiplex PCR assay showing lysogeny during a phage infection time course in the strain lacking BREX (black), but not in BREX‐containing strain (red), or uninfected (U) strains. Amplicons for the bacterial DNA, phage DNA, and lysogen‐specific DNA are 293, 485, and 1,218 bp, respectively.


Figure 3A BREX does not work via an abortive infection mechanism . Increasing the MOI of the Φ3T infection from 0 . 05 to 5 shortens the time to culture crash for the BREX ‐ lacking strain , but does not result in culture decline for BREX ‐ containing strain . Error bars represent SD of technical triplicates . B The system does not interfere with phage adsorption to bacterial cells . Strains either containing ( red ) or lacking ( black ) the BREX system were infected with phage Φ3T and then chloroform ‐ treated 15 min following infection . The culture was plated on Φ3T ‐ sensitive B . subtilis cells and plaques , representing extracellular , unadsorbed phages , were counted . C Phage DNA replication does not occur in BREX ‐ containing cells ( red ) , but is observed in the BREX ‐ lacking strain ( black ) . Y ‐ axis represents relative phage concentrations normalized to the value at the beginning of the infection , as measured by Illumina sequencing .

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

Figure 3A BREX does not work via an abortive infection mechanism. Increasing the MOI of the Φ3T infection from 0.05 to 5 shortens the time to culture crash for the BREX‐lacking strain, but does not result in culture decline for BREX‐containing strain. Error bars represent SD of technical triplicates.B The system does not interfere with phage adsorption to bacterial cells. Strains either containing (red) or lacking (black) the BREX system were infected with phage Φ3T and then chloroform‐treated 15 min following infection. The culture was plated on Φ3T‐sensitive B. subtilis cells and plaques, representing extracellular, unadsorbed phages, were counted.C Phage DNA replication does not occur in BREX‐containing cells (red), but is observed in the BREX‐lacking strain (black). Y‐axis represents relative phage concentrations normalized to the value at the beginning of the infection, as measured by Illumina sequencing.


Figure 4C , D Culture dynamics of non ‐ infected ( C ) or phage ‐ infected ( D ) cultures . Curves depict culture dynamics of strains lacking BREX ( black ) and BREX ‐ containing ( red ) strains of B . subtilis BEST7003 , as well as a BREX ‐ containing strain where the pglX methylase was deleted ( green ) . Axes and error bars are as in Fig . E Southern blot analysis of phage Φ3T genome during infection . Numbers indicate time ( in min ) following infection ; U , uninfected . Probe was designed to match positions 94 , 645 - 95 , 416 in the phage genome . Each lane contains 200 ng total DNA .

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

Figure 4C, D Culture dynamics of non‐infected (C) or phage‐infected (D) cultures. Curves depict culture dynamics of strains lacking BREX (black) and BREX‐containing (red) strains of B. subtilis BEST7003, as well as a BREX‐containing strain where the pglX methylase was deleted (green). Axes and error bars are as in Fig .E Southern blot analysis of phage Φ3T genome during infection. Numbers indicate time (in min) following infection; U, uninfected. Probe was designed to match positions 94,645-95,416 in the phage genome. Each lane contains 200 ng total DNA.


Figure 2 ( A ) MCF - 7 cells were co - transfected with either MIR - CNT ( control plasmid ) or MIR376A and GFP - LC3 plasmid , and GFP - LC3 dot formation was analyzed . White arrows indicate clusters of the GFP - LC3 dots in cells . ( B ) Quantification of the experiments in A . MIR376A overexpression , but not MIR - CNT expression , blocked starvation ( STV ) - induced autophagy ( mean ± S . D . of independent experiments , n = 4 , * * * p < 0 , 01 ) . NON STV , non - starved ( C ) Overexpression of MIR376A resulted in a decrease in the autophagic activity of MCF - 7 cells . Starvation - induced conversion of LC3 - I to LC3 - II in MCF - 7 cells was analyzed . Tests were performed in the presence or absence of E64d ( 10 µg / ml ) and Pepstatin A ( 10 µg / ml ) ( E + P ) . LC3 - II / LC3 - I densitometric ratios were marked . ACTB was used as a loading control . ( D ) MIR376A blocked starvation induced SQSTM1 degradation in MCF - 7 cells . ACTB was used as a loading control . SQSTM1 / ACTIN densitometric ratios were marked .

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

Figure 2(A) MCF-7 cells were co-transfected with either MIR-CNT (control plasmid) or MIR376A and GFP-LC3 plasmid, and GFP-LC3 dot formation was analyzed. White arrows indicate clusters of the GFP-LC3 dots in cells. (B) Quantification of the experiments in A. MIR376A overexpression, but not MIR-CNT expression, blocked starvation (STV)-induced autophagy (mean ± S.D. of independent experiments, n = 4, ***p<0,01). NON STV, non-starved(C) Overexpression of MIR376A resulted in a decrease in the autophagic activity of MCF-7 cells. Starvation-induced conversion of LC3-I to LC3-II in MCF-7 cells was analyzed. Tests were performed in the presence or absence of E64d (10 µg/ml) and Pepstatin A (10 µg/ml) (E+P). LC3-II/LC3-I densitometric ratios were marked. ACTB was used as a loading control.(D) MIR376A blocked starvation induced SQSTM1 degradation in MCF-7 cells. ACTB was used as a loading control. SQSTM1/ACTIN densitometric ratios were marked.


Figure 3 ( A ) MIR376A blocked GFP - LC3 dot formation under starvation condition . ( A ) MIR376A blocked GFP - LC3 dot formation under starvation condition . ( B ) Quantitative analysis of experiments in A ( mean ± S . D . of independent experiments , n = 3 , * * * p < 0 , 01 ) . ( C ) Overexpression of MIR376A resulted in decreased autophagic flux in Huh - 7 cells . Starvation - induced conversion of LC3 - I to LC3 - II was analyzed . Tests were performed in the presence or absence of E64d ( 10 µg / ml ) and Pepstatin A ( 10 µg / ml ) ( E + P ) . LC3 - II / LC3 - I densitometric ratios were marked . ACTB was used as a loading control .

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

Figure 3(A) MIR376A blocked GFP-LC3 dot formation under starvation condition.(A) MIR376A blocked GFP-LC3 dot formation under starvation condition. (B) Quantitative analysis of experiments in A (mean ± S.D. of independent experiments, n = 3, ***p<0,01).(C) Overexpression of MIR376A resulted in decreased autophagic flux in Huh-7 cells. Starvation-induced conversion of LC3-I to LC3-II was analyzed. Tests were performed in the presence or absence of E64d (10 µg/ml) and Pepstatin A (10 µg/ml) (E+P). LC3-II/LC3-I densitometric ratios were marked. ACTB was used as a loading control.


Figure 4 ( C and D ) qPCR analysis of ATG4C and BECN1 mRNA expression in control miRNA ( MIR - CONT ) or MIR376A overexpressing cells under non - starved ( NON STV ) or starved ( STV ) condition ( mean ± S . D . of independent experiments , n = 3 , * p < 0 , 05 ) . ( E and F ) Immunoblots showing ATG4C and BECN1 protein levels following control miRNA ( MIR - CONT ) or MIR376A overexpression . ACTB was used as a loading control . ATG4C / ACTB or BECN1 / ACTB densitometric ratios were marked .

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

Figure 4(C and D) qPCR analysis of ATG4C and BECN1 mRNA expression in control miRNA (MIR-CONT) or MIR376A overexpressing cells under non-starved (NON STV) or starved (STV) condition (mean ± S.D. of independent experiments, n = 3, *p<0,05).(E and F) Immunoblots showing ATG4C and BECN1 protein levels following control miRNA (MIR-CONT) or MIR376A overexpression. ACTB was used as a loading control. ATG4C/ACTB or BECN1/ACTB densitometric ratios were marked.


Figure 5 ( A and B ) qPCR analysis of ATG4C and BECN1 mRNA levels in MCF - 7 cells transfected with control antagomirs ( CNT - Ant ) or antagomir - 376a ( Ant - 376a ) ( mean ± S . D . of independent experiments , n = 3 , * * p < 0 , 03 for ATG4C , and n = 3 , * * p < 0 , 03 for BECN1 ) . Results were expressed as fold changes against GAPDH mRNA levels . ( C and D ) ATG4C and BECN1 protein levels were increased following antagomir - 376a ( Ant - 376a ) transfection . ATG4C / ACTB or BECN1 / ACTB densitometric ratios were marked .

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

Figure 5(A and B) qPCR analysis of ATG4C and BECN1 mRNA levels in MCF-7 cells transfected with control antagomirs (CNT-Ant) or antagomir-376a (Ant-376a) (mean ± S.D. of independent experiments, n = 3, **p<0,03 for ATG4C, and n = 3, **p<0,03 for BECN1). Results were expressed as fold changes against GAPDH mRNA levels.(C and D) ATG4C and BECN1 protein levels were increased following antagomir-376a (Ant-376a) transfection. ATG4C/ACTB or BECN1/ACTB densitometric ratios were marked.


Figure 6 ( C and D ) Normalized luciferase activity measuremets of lysates from 293T cells that were co - transfected with either MIR - CNT or MIR376A plasmids together with the wild - type or mutant luciferase constructs ( mean ± S . D . of independent experiments , n = 3 for ATG4C tests , * p < 0 , 05 and n = 5 for BECN1 tests , * * * p < 0 , 01 ) .

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

Figure 6(C and D) Normalized luciferase activity measuremets of lysates from 293T cells that were co-transfected with either MIR-CNT or MIR376A plasmids together with the wild-type or mutant luciferase constructs (mean ± S.D. of independent experiments, n = 3 for ATG4C tests, *p<0,05 and n = 5 for BECN1 tests, ***p<0,01).


Figure 7 ( A ) Blockage of endogenous MIR376A by Ant - 376a , but not CNT - Ant further stimulated starvation ( STV ) - activated LC3 - I to LC3 - II conversion in MCF - 7 cells . ACTB was used as a loading control . LC3 - II / LC3 - I densitometric ratios were marked . ( B ) Ant - 376a , but not CNT - Ant resulted in further activation of SQSTM1 protein degradation following starvation in MCF - 7 cells . SQSTM1 / ACTB ratios were marked .

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

Figure 7(A) Blockage of endogenous MIR376A by Ant-376a, but not CNT-Ant further stimulated starvation (STV)-activated LC3-I to LC3-II conversion in MCF-7 cells. ACTB was used as a loading control. LC3-II/LC3-I densitometric ratios were marked.(B) Ant-376a, but not CNT-Ant resulted in further activation of SQSTM1 protein degradation following starvation in MCF-7 cells. SQSTM1/ACTB ratios were marked.


Figure 8 MIR376A and MIR376B level changes in response to starvation stress . ( A and B ) TaqMan quantitative PCR ( qPCR ) analysis of endogenous MIR376A ( A ) and MIR376B ( B ) levels under control ( CNT , no starvation ) or starvation ( STV , 1 to 8 hours ) conditions . TaqMan qPCR data was normalized using U6 small nuclear 1 ( RNU6 - 1 ) ( U6 ) mRNA levels ( mean ± S . D . of independent experiments , n = 5 for both A and B , * * * p < 0 , 01 ) .

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

Figure 8 MIR376A and MIR376B level changes in response to starvation stress. (A and B) TaqMan quantitative PCR (qPCR) analysis of endogenous MIR376A (A) and MIR376B (B) levels under control (CNT, no starvation) or starvation (STV, 1 to 8 hours) conditions. TaqMan qPCR data was normalized using U6 small nuclear 1 (RNU6-1) (U6) mRNA levels (mean ± S.D. of independent experiments, n = 5 for both A and B, ***p<0,01).


Figure 4C . Dissociation constants ( Kd ) of Ago2 - miRNA complexes ( containing an AU - rich supplementary region ) binding to target RNAs with bridging regions of various lengths . D . Dissociation constants ( Kd ) of Ago2 - miRNA complexes ( containing an GC - rich supplementary region ) binding to target RNAs with bridging regions of various lengths . Data information : All plotted data are the means of at least three independent replicates . Error bars indicate SEM . E . Fold repression of a luciferase reporter ( relative to no target site control ) plotted versus affinity ( 1 / Kd ) values measured for the same sites in vitro . Data information : All plotted data are the means of at least three independent replicates . Error bars indicate SEM .

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Figure 4C. Dissociation constants (Kd) of Ago2-miRNA complexes (containing an AU-rich supplementary region) binding to target RNAs with bridging regions of various lengths. D. Dissociation constants (Kd) of Ago2-miRNA complexes (containing an GC-rich supplementary region) binding to target RNAs with bridging regions of various lengths. Data information: All plotted data are the means of at least three independent replicates. Error bars indicate SEM.E. Fold repression of a luciferase reporter (relative to no target site control) plotted versus affinity (1/Kd) values measured for the same sites in vitro. Data information: All plotted data are the means of at least three independent replicates. Error bars indicate SEM.


Figure 5A . Top : schematic of miR - 122 isomiRs paired with a target complementary to the seed region only . Bottom : release of the seed - paired target from Ago2 - miR complexes was assessed by monitoring the fraction of the total target RNA bound as a function of time . B . Top : schematic of miR - 122 isomiRs paired with a target complementary to the seed and supplementary ( g13 - g17 ) regions . Bottom : fraction target bound to the Ago2 - miR complexes as a function of time . C . Top : schematic of miR - 122 isomiRs paired with a target complementary to the seed and shifted supplementary ( g15 - g19 ) regions . . Bottom : fraction target bound to the Ago2 - miR complexes as a function of time . Data information : All plotted data are the means of at least three independent replicates . Error bars indicate SEM . E . Release half times ( t1 / 2 ) of target RNAs in panels ( A - C ) plotted against isomiR length . Data information : All plotted data are the means of at least three independent replicates . Error bars indicate SEM .

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

Figure 5A. Top: schematic of miR-122 isomiRs paired with a target complementary to the seed region only. Bottom: release of the seed-paired target from Ago2-miR complexes was assessed by monitoring the fraction of the total target RNA bound as a function of time. B. Top: schematic of miR-122 isomiRs paired with a target complementary to the seed and supplementary (g13-g17) regions. Bottom: fraction target bound to the Ago2-miR complexes as a function of time. C. Top: schematic of miR-122 isomiRs paired with a target complementary to the seed and shifted supplementary (g15-g19) regions. . Bottom: fraction target bound to the Ago2-miR complexes as a function of time. Data information: All plotted data are the means of at least three independent replicates. Error bars indicate SEM.E. Release half times (t1/2) of target RNAs in panels (A-C) plotted against isomiR length. Data information: All plotted data are the means of at least three independent replicates. Error bars indicate SEM.


Figure 1Overall structure of the RBD - ACE2 - B0AT1 complex . ( A ) Cryo - EM map of the RBD - ACE2 - B0AT1 complex . Left : Overall reconstruction of the ternary complex at 2 . 9 Å . Inset : focused refined map of RBD . Overall structure of the RBD - ACE2 - B0AT1 complex . The complex is colored by subunits , with the protease domain ( PD ) and the Collectrin - like domain ( CLD ) colored cyan and blue in one of the ACE2 protomers , respectively . The glycosylation moieties are shown as sticks .

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

Figure 1Overall structure of the RBD-ACE2-B0AT1 complex.(A) Cryo-EM map of the RBD-ACE2-B0AT1 complex. Left: Overall reconstruction of the ternary complex at 2.9 Å. Inset: focused refined map of RBD.Overall structure of the RBD-ACE2-B0AT1 complex. The complex is colored by subunits, with the protease domain (PD) and the Collectrin-like domain (CLD) colored cyan and blue in one of the ACE2 protomers, respectively. The glycosylation moieties are shown as sticks.


Figure 2 Cryo - EM density of the interface between RBD and ACE2 . The density , shown as pink meshes , is contoured at 12 σ .

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Figure 2 Cryo-EM density of the interface between RBD and ACE2.The density, shown as pink meshes, is contoured at 12 σ.