( G ) Cytokine production by CD19 + HLA - DR + B cells and CD11c + dendritic cells in the presence of increasing doses ( 15 - 1 , 000 nM ) of BTP2 . Left : Dose - response curves showing frequencies of cytokine - producing cells after 4h stimulation with PMA / ionomycin ex vivo . Data are normalized to control samples stimulated with PMA / ionomycin in the absence of BTP2 . Error bars represent SEM obtained from 1 experiment ( n = 5 CD patients ) . Right : heatmaps showing the median fold change of cytokines and surface markers in CD19 + HLA - DR + B cells and CD11c + dendritic cells treated as described for the left panel .

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

(G) Cytokine production by CD19+HLA-DR+ B cells and CD11c+ dendritic cells in the presence of increasing doses (15-1,000 nM) of BTP2. Left: Dose-response curves showing frequencies of cytokine-producing cells after 4h stimulation with PMA/ionomycin ex vivo. Data are normalized to control samples stimulated with PMA/ionomycin in the absence of BTP2. Error bars represent SEM obtained from 1 experiment (n=5 CD patients). Right: heatmaps showing the median fold change of cytokines and surface markers in CD19+HLA-DR+ B cells and CD11c+ dendritic cells treated as described for the left panel.


( A ) Experimental setup for mass cytometric assays . Non - inflamed : n = 4 , CD : n = 6 , UC : n = 6 . ( B ) FlowSOM plot of merged FCS files from LPMCs of non - inflamed controls , UC or CD patients after stimulation with PMA / ionomycin ± 1 μM BTP2 for 4h ex vivo . Colors of the t - SNE plot represent 20 clusters of distinct CD45 + LPMC lineages . The heatmap shows the expression levels of 21 markers used for defining cell clusters .

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

(A) Experimental setup for mass cytometric assays. Non-inflamed: n = 4, CD: n =6, UC: n =6. (B) FlowSOM plot of merged FCS files from LPMCs of non-inflamed controls, UC or CD patients after stimulation with PMA/ionomycin ± 1 μM BTP2 for 4h ex vivo. Colors of the t-SNE plot represent 20 clusters of distinct CD45+ LPMC lineages. The heatmap shows the expression levels of 21 markers used for defining cell clusters.


( C ) FlowSOM map of merged FCS files from CD45 + cells of non - inflamed controls , UC and CD patients that were left unstimulated or treated with PMA / ionomycin ± 1 μM BTP2 . Colors indicate altered cell clusters in CD patients vs . controls , UC patients vs . controls and CD vs . UC patients . ( D ) viSNE plots of one exemplary non - inflamed control , UC and CD patient colored by marker expression levels ( blue : low , red : high ) .

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

(C) FlowSOM map of merged FCS files from CD45+ cells of non-inflamed controls, UC and CD patients that were left unstimulated or treated with PMA/ionomycin ± 1 μM BTP2. Colors indicate altered cell clusters in CD patients vs. controls, UC patients vs. controls and CD vs. UC patients. (D) viSNE plots of one exemplary non-inflamed control, UC and CD patient colored by marker expression levels (blue: low, red: high).


( E ) Quantified frequencies ( % ) of each cell subset defined by the cluster analysis . Statistical significance was calculated using the edgeR statistical framework with negative binomial GLM and a false discovery rate adjusted to 10 % using the Benjiamini - Hochberg procedure ( * padj < 0 . 05 , * * padj < 0 . 01 , * * * padj < 0 . 001 ) . Boxes extend from the 25th to 75th percentiles . Whisker plots show the min ( smallest ) and max ( largest ) values . The line in the box denotes the median from 1 experiment ( non - inflamed n = 4 , CD : n = 6 , UC : n = 6 patients ) .

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

(E) Quantified frequencies (%) of each cell subset defined by the cluster analysis. Statistical significance was calculated using the edgeR statistical framework with negative binomial GLM and a false discovery rate adjusted to 10% using the Benjiamini-Hochberg procedure (*padj< 0.05, **padj < 0.01, ***padj< 0.001). Boxes extend from the 25th to 75th percentiles. Whisker plots show the min (smallest) and max (largest) values. The line in the box denotes the median from 1 experiment (non-inflamed n = 4, CD: n =6, UC: n =6 patients).


( F ) Heatmaps representing the median fold change of cytokine and surface marker expression in CD45 + LPMCs that were isolated from non - inflamed , UC and CD samples and activated for 4h with PMA / ionomycin ex vivo . Statistical significance was calculated using an unpaired t test with FDR adjustment to 1 % using the Benjamini , Krieger and Yekutieli procedure ; *

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

(F) Heatmaps representing the median fold change of cytokine and surface marker expression in CD45+ LPMCs that were isolated from non-inflamed, UC and CD samples and activated for 4h with PMA/ionomycin ex vivo. Statistical significance was calculated using an unpaired t test with FDR adjustment to 1% using the Benjamini, Krieger and Yekutieli procedure; *


( A ) FlowSOM plot of merged FCS files from samples treated with PMA / ionomycin ± 1 µM BTP2 ( non - inflamed : n = 4 , CD : n = 6 , UC : n = 6 ) . Colors of FlowSOM plot indicate 20 distinct clusters of CD45 + CD3 + LPMCs . Heatmaps show the expression levels of 24 markers used for cluster determination . The table shows the mean ± SEM of frequencies ( % ) for each cell subset defined in non - inflamed controls , UC and CD patients after 4h stimulation with PMA / ionomycin in vitro . Statistical significance was calculated using the edgeR statistical framework with negative binomial GLM and a false discovery rate adjusted to 10 % using the Benjiamini - Hochberg procedure .

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

(A) FlowSOM plot of merged FCS files from samples treated with PMA/ionomycin ± 1 µM BTP2 (non-inflamed: n = 4, CD: n =6, UC: n =6). Colors of FlowSOM plot indicate 20 distinct clusters of CD45+CD3+ LPMCs. Heatmaps show the expression levels of 24 markers used for cluster determination. The table shows the mean ± SEM of frequencies (%) for each cell subset defined in non-inflamed controls, UC and CD patients after 4h stimulation with PMA/ionomycin in vitro. Statistical significance was calculated using the edgeR statistical framework with negative binomial GLM and a false discovery rate adjusted to 10% using the Benjiamini-Hochberg procedure.


( B ) Box plots showing frequencies ( % ) of each cell subset defined by the cluster analysis in non - inflamed , UC and CD samples following stimulation with PMA / ionomycin ± BTP2 for 4h in vitro . Statistical significances were calculated using a one - tailed paired Wilcoxon matched - pairs signed - rank test , * p < 0 . 05 . Boxes range from the 25th to 75th percentiles . Whisker plots show the min ( smallest ) and max ( largest ) values . The line in the box denotes the median obtained from 1 experiment ( non - inflamed : n = 4 , CD : n = 6 , UC : n = 6 patients ) .

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

(B) Box plots showing frequencies (%) of each cell subset defined by the cluster analysis in non-inflamed, UC and CD samples following stimulation with PMA/ionomycin ± BTP2 for 4h in vitro. Statistical significances were calculated using a one-tailed paired Wilcoxon matched-pairs signed-rank test, *p < 0.05. Boxes range from the 25th to 75th percentiles. Whisker plots show the min (smallest) and max (largest) values. The line in the box denotes the median obtained from 1 experiment (non-inflamed: n = 4, CD: n =6, UC: n =6 patients).


( C ) viSNE plots of one exemplary CD patient . Identification of cell subsets ( left ) and expression levels of cytokines after PMA / ionomycin stimulation in the presence or absence of BTP2 ( blue ; low , red : high ) .

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

(C) viSNE plots of one exemplary CD patient. Identification of cell subsets (left) and expression levels of cytokines after PMA/ionomycin stimulation in the presence or absence of BTP2 (blue; low, red: high).


( D ) Heatmaps indicating the median fold change expression of cytokines and surface molecules in CD45 + CD3 + LPMCs stimulated with PMA / ionomycin ± BTP2 for 4h in vitro . Values are normalized to stimulated , non - BTP2 treated samples .

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

(D) Heatmaps indicating the median fold change expression of cytokines and surface molecules in CD45+CD3+ LPMCs stimulated with PMA/ionomycin ± BTP2 for 4h in vitro. Values are normalized to stimulated, non-BTP2 treated samples.


( A ) FlowSOM plot of merged FCS files from unstimulated samples or samples treated with PMA / ionomycin ± 1 µM BTP2 ( non - inflamed : n = 4 , CD : n = 6 , UC : n = 6 ) . Colors of FlowSOM plot indicate 20 distinct clusters of CD45 + CD3 - LPMCs . Heatmap clusters showing the expression levels of 34 markers used for cluster determination . The table shows the mean ± SEM of frequencies ( % ) for each cell subset defined in non - Inflamed , UC and CD samples after stimulation with PMA / ionomycin for 4h . Statistical significance was calculated using the edgeR statistical framework with negative binomial GLM and a false discovery rate adjusted to 10 % using the Benjiamini - Hochberg

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

(A) FlowSOM plot of merged FCS files from unstimulated samples or samples treated with PMA/ionomycin ± 1 µM BTP2 (non-inflamed: n = 4, CD: n =6, UC: n =6). Colors of FlowSOM plot indicate 20 distinct clusters of CD45+CD3- LPMCs. Heatmap clusters showing the expression levels of 34 markers used for cluster determination. The table shows the mean ± SEM of frequencies (%) for each cell subset defined in non-Inflamed, UC and CD samples after stimulation with PMA/ionomycin for 4h. Statistical significance was calculated using the edgeR statistical framework with negative binomial GLM and a false discovery rate adjusted to 10% using the Benjiamini-Hochberg procedure


( B ) Box plots show frequencies ( % ) of each cell subset defined by the cluster analysis in samples of non - inflamed controls , UC or CD patients after stimulation with PMA / ionomycin ± BTP2 for 4h ex vivo . Statistical significances were calculated by one - tailed paired Wilcoxon matched - pairs signed - rank test , * p < 0 . 05 . Boxes range from the 25th to 75th percentiles . Whisker plots show the min ( smallest ) and max ( largest ) values . The line in the box denotes the median obtained from 1 experiment ( non - inflamed : n = 4 , CD : n = 6 , UC : n = 6 patients ) .

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

(B) Box plots show frequencies (%) of each cell subset defined by the cluster analysis in samples of non-inflamed controls, UC or CD patients after stimulation with PMA/ionomycin ± BTP2 for 4h ex vivo. Statistical significances were calculated by one-tailed paired Wilcoxon matched-pairs signed-rank test, *p < 0.05. Boxes range from the 25th to 75th percentiles. Whisker plots show the min (smallest) and max (largest) values. The line in the box denotes the median obtained from 1 experiment (non-inflamed: n = 4, CD: n =6, UC: n =6 patients).


( C ) viSNE plots of one exemplary CD patient . Identification of cell subsets ( left ) and expression levels of cell markers after PMA / ionomycin stimulation in the presence or absence of BTP2 ( blue ; low , red : high ) .

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

(C) viSNE plots of one exemplary CD patient. Identification of cell subsets (left) and expression levels of cell markers after PMA/ionomycin stimulation in the presence or absence of BTP2 (blue; low, red: high).


( D ) Heatmaps indicating the median fold change expression of cytokines and surface markers in CD45 + CD3 - LPMCs activated with PMA / ionomycin ± BTP2 for 4h in vitro . Values are normalized to stimulated , non - BTP2 treated samples .

(D) Heatmaps indicating the median fold change expression of cytokines and surface markers in CD45+CD3- LPMCs activated with PMA/ionomycin ± BTP2 for 4h in vitro. Values are normalized to stimulated, non-BTP2 treated samples.


( A - B ) Adult stem cell - derived intestinal epithelial spheroids of wild - type C57BL / 6 mice were cultured for 4 days in Wnt - free medium to induce differentiation . Initial viability was determined on day 0 . DMSO control ( vehicle ) or 1 μM BTP2 were added to the culture medium and viability was measured every 24h by resazurin viability assay and microscopy . Scale bar depicts 100 µm . ( C ) Transepithelial electrical resistance ( TEER ) was measured on differentiated murine colon monolayers in the presence or absence of 1 µM BTP2 .

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

(A-B) Adult stem cell-derived intestinal epithelial spheroids of wild-type C57BL/6 mice were cultured for 4 days in Wnt-free medium to induce differentiation. Initial viability was determined on day 0. DMSO control (vehicle) or 1 μM BTP2 were added to the culture medium and viability was measured every 24h by resazurin viability assay and microscopy. Scale bar depicts 100 µm. (C) Transepithelial electrical resistance (TEER) was measured on differentiated murine colon monolayers in the presence or absence of 1 µM BTP2.


( D ) Human colonic spheroids were generated from primary human epithelial crypts obtained from colon resectates of 2 CD patients and cultured for 6 days in Wnt - free medium to induce differentiation into colonic organoids . Initial viability was determined on day 0 . DMSO control or 1 μM BTP2 were added to the culture medium and viability was measured on days 2 , 4 and 6 .

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

(D) Human colonic spheroids were generated from primary human epithelial crypts obtained from colon resectates of 2 CD patients and cultured for 6 days in Wnt-free medium to induce differentiation into colonic organoids. Initial viability was determined on day 0. DMSO control or 1 μM BTP2 were added to the culture medium and viability was measured on days 2, 4 and 6.


( E ) TEER of differentiated human colon monolayers was analyzed in the presence or absence of 1 µM BTP2 .

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

(E) TEER of differentiated human colon monolayers was analyzed in the presence or absence of 1 µM BTP2.


( F ) Relative mRNA expression of markers for enterocytes ( ALPI ) , goblet cells ( MUC2 ) , and entero - endocrine cells ( CHGA ) was quantified by RT - PCR in differentiated human colon monolayers after addition of 1 µM BTP2 .

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

(F) Relative mRNA expression of markers for enterocytes (ALPI), goblet cells (MUC2), and entero-endocrine cells (CHGA) was quantified by RT-PCR in differentiated human colon monolayers after addition of 1 µM BTP2.


( A - C ) Acute administration of the CRAC channel blocker CM4620 inhibits CRAC currents ( Icrac ) . ( A ) Time course of peak amplitudes of ICRAC before and after application of 3 μM CM4620 to HEK293 cells transfected with ORAI1 and STIM1 . ( B ) Current - voltage ( I - V ) relationship of ICRAC before and after CM4620 treatment . I - Vs were obtained at the time points indicated by the arrowheads in ( A ) . ( C ) Fractional blockade of ICRAC by CM4620 was measured by comparing current amplitudes before and after CM4620 administration ( arrowheads ) using the formula 1 - ( ICM4620 / ICtrl ) . Data are from 8 cells per conditions and shown as the mean ± SEM .

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

(A-C) Acute administration of the CRAC channel blocker CM4620 inhibits CRAC currents (Icrac). (A) Time course of peak amplitudes of ICRAC before and after application of 3 μM CM4620 to HEK293 cells transfected with ORAI1 and STIM1. (B) Current-voltage (I-V) relationship of ICRAC before and after CM4620 treatment. I-Vs were obtained at the time points indicated by the arrowheads in (A). (C) Fractional blockade of ICRAC by CM4620 was measured by comparing current amplitudes before and after CM4620 administration (arrowheads) using the formula 1- (ICM4620 / ICtrl). Data are from 8 cells per conditions and shown as the mean ± SEM.


( E ) Relative weight loss of mice treated with vehicle or CM4620 ; the start of treatment is indicated by the white arrow . Data are the mean ± SEM of 9 mice per cohort , statistical analysis by unpaired student ' s t - test , * p < 0 . 05 .

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(E) Relative weight loss of mice treated with vehicle or CM4620; the start of treatment is indicated by the white arrow. Data are the mean ± SEM of 9 mice per cohort, statistical analysis by unpaired student's t-test , *p<0.05.


( F ) Concentrations of CM4620 in the serum of mice at day 49 , data are the mean ± SEM . Statistical analysis by unpaired student ' s t - test , * * * p < 0 . 001 .

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(F) Concentrations of CM4620 in the serum of mice at day 49, data are the mean ± SEM. Statistical analysis by unpaired student's t-test, ***p<0.001.


( G ) Representative H & E staining of the distal colon of Rag1 - / - mice treated with vehicle or CM4620 . Colitis scores of 9 mice per cohort . Each symbol represents one mouse .

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(G) Representative H&E staining of the distal colon of Rag1-/- mice treated with vehicle or CM4620. Colitis scores of 9 mice per cohort. Each symbol represents one mouse.


( H ) Number of CD11b + Gr - 1 + neutrophils and CD4 + T cells ( normalized to grams of tissue ) in the colon lamina propria ( CLP ) of mice on day 49 quantified by flow cytometry . Each dot represents one mouse . Data are the mean ± SEM of 9 mice per cohort .

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

(H) Number of CD11b+Gr-1+ neutrophils and CD4+ T cells (normalized to grams of tissue) in the colon lamina propria (CLP) of mice on day 49 quantified by flow cytometry. Each dot represents one mouse. Data are the mean ± SEM of 9 mice per cohort.


( I ) Frequencies of RORγt + Th17 cells and Foxp3 + Treg cells in the CLP of mice treated with CM4620 or vehicle . Each dot represents one mouse . Data are the mean ± SEM of 6 control and 5 CM4620 treated mice .

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

(I) Frequencies of RORγt+ Th17 cells and Foxp3+ Treg cells in the CLP of mice treated with CM4620 or vehicle. Each dot represents one mouse. Data are the mean ± SEM of 6 control and 5 CM4620 treated mice.


( J ) Frequencies of IFN - γ , TNF - α and IL - 2 producing CD4 + T cells isolated from the CLP , restimulated ex vivo with PMA and ionomycin for 4 h ( without addition of CM4620 during stimulation in vitro ) and analyzed by flow cytometry . Bar graphs represent the percentages of IFN - γ + , TNF - α + , IL - 2 + cells in CD4 + cells ( top row ) and mean fluorescent intensity ( MFI ) of IFN - γ , TNF - α , IL - 2 on CD4 + cells ( bottom row ) . Each dot represents one mouse . Data are the mean ± SEM of 6 control and 5 CM4620 treated mice .

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

(J) Frequencies of IFN-γ, TNF-α and IL-2 producing CD4+ T cells isolated from the CLP, restimulated ex vivo with PMA and ionomycin for 4 h (without addition of CM4620 during stimulation in vitro) and analyzed by flow cytometry. Bar graphs represent the percentages of IFN-γ+, TNF-α+, IL-2+ cells in CD4+ cells (top row) and mean fluorescent intensity (MFI) of IFN-γ, TNF-α, IL-2 on CD4+ cells (bottom row). Each dot represents one mouse. Data are the mean ± SEM of 6 control and 5 CM4620 treated mice.


Neurosphere assays were performed using mouse E14 . 5 primary neural progenitors transduced with a retroviral vector expressing

Neurosphere assays were performed using mouse E14.5 primary neural progenitors transduced with a retroviral vector expressing Ttyh1


XTT assays were performed using mouse E14 . 5 primary neural progenitors transduced with a retroviral vector expressing

XTT assays were performed using mouse E14.5 primary neural progenitors transduced with a retroviral vector expressing Ttyh1


Neurosphere assays were performed using mouse E14 . 5 primary neural progenitors transduced with a short hairpin RNA specific to Ttyh1 ( shTtyh1 )

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

Neurosphere assays were performed using mouse E14.5 primary neural progenitors transduced with a short hairpin RNA specific to Ttyh1 (shTtyh1)


XTT assays were performed using mouse E14 . 5 primary neural progenitors transduced with a short hairpin RNA specific to Ttyh1 ( shTtyh1 )

XTT assays were performed using mouse E14.5 primary neural progenitors transduced with a short hairpin RNA specific to Ttyh1 (shTtyh1)


Ttyh1 - expressing cells in E15 . 5 embryonic brains that were intraventricularly electroporated at E13 . 5 were immunostained using an anti - GFP primary ( reporter gene ) and Alexa Fluor 488 - conjugated secondary antibodies . GFP immunofluorescence ( green ) was merged with DAPI - counterstained images ( blue ) . VZ , ventricular zone ; SVZ , subventricular zone ; IZ , intermediate zone ; CP , cortical plate . Scale bar : 100 μm . Quantification of ( E )

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

Ttyh1-expressing cells in E15.5 embryonic brains that were intraventricularly electroporated at E13.5 were immunostained using an anti-GFP primary (reporter gene) and Alexa Fluor 488-conjugated secondary antibodies. GFP immunofluorescence (green) was merged with DAPI-counterstained images (blue). VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bar: 100 μm. Quantification of (E)


Double - immunolabeling of E15 . 5 brain sections electroporated in utero with Ttyh1 - expressing plasmid at E13 . 5 using anti - GFP ( green ) and Sox2 ( red ) antibodies , and Alexa Fluor 488 - and 555 - conjugated secondary antibodies . Scale bar : 50 μm .

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

Double-immunolabeling of E15.5 brain sections electroporated in utero with Ttyh1-expressing plasmid at E13.5 using anti-GFP (green) and Sox2 (red) antibodies, and Alexa Fluor 488- and 555-conjugated secondary antibodies. Scale bar: 50 μm.


Double - immunolabeling of E15 . 5 brain sections electroporated in utero with Ttyh1 - expressing plasmid at E13 . 5 using anti - GFP ( green ) and Sox2 ( red ) antibodies , and Alexa Fluor 488 - and 555 - conjugated secondary antibodies . Scale bar : 50 μm . Quantification of ( G ) ( n = 3 ) .

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

Double-immunolabeling of E15.5 brain sections electroporated in utero with Ttyh1-expressing plasmid at E13.5 using anti-GFP (green) and Sox2 (red) antibodies, and Alexa Fluor 488- and 555-conjugated secondary antibodies. Scale bar: 50 μm. Quantification of (G) (n = 3).


Immunolabeling of E14 . 5 brain sections electroporated in utero with shTtyh1 alone or in combination with a shTtyh1 - resistant Ttyh1 ( Ttyh1res ) expression vector at E13 . 5 using anti - GFP and Alexa Fluor 488 - conjugated secondary antibodies . The DAPI nuclear counterstain is shown in blue . Scale bar : 100 μm . Quantification of ( I )

Immunolabeling of E14.5 brain sections electroporated in utero with shTtyh1 alone or in combination with a shTtyh1-resistant Ttyh1 (Ttyh1res) expression vector at E13.5 using anti-GFP and Alexa Fluor 488-conjugated secondary antibodies. The DAPI nuclear counterstain is shown in blue. Scale bar: 100 μm. Quantification of (I)


Two days after E14 . 5 primary neural progenitor cells were transduced with retroviral vectors expressing ( A ) Ttyh1 mRNA expression levels of each indicated Notch target gene were measured by

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

Two days after E14.5 primary neural progenitor cells were transduced with retroviral vectors expressing (A) Ttyh1 mRNA expression levels of each indicated Notch target gene were measured by qPCR


Two days after E14 . 5 primary neural progenitor cells were transduced with shTtyh1 , mRNA expression levels of each indicated Notch target gene were measured by

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Two days after E14.5 primary neural progenitor cells were transduced with shTtyh1, mRNA expression levels of each indicated Notch target gene were measured by qPCR


Effects of dominant negative MAML1 ( dnMAML1 ) on Ttyh1 - enhanced Notch target gene expression were tested by qPCR using E14 . 5 primary neural progenitors at 2 days posttransduction ( n = 4 ) .

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Effects of dominant negative MAML1 (dnMAML1) on Ttyh1-enhanced Notch target gene expression were tested by qPCR using E14.5 primary neural progenitors at 2 days posttransduction (n = 4).


The dnMAML1 plasmid was introduced with the Ttyh1 vector into embryonic brains at E13 . 5 and cell localization was analyzed at E15 . 5 by anti - GFP immunostaining . GFP and DAPI signals are shown in green and blue , respectively . VZ , ventricular zone ; SVZ , subventricular zone ; IZ , intermediate zone ; CP , cortical plate . Scale bar : 100 μm . E Quantification of GFP + cell positions in ( D ) ( n = 3 ) .

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The dnMAML1 plasmid was introduced with the Ttyh1 vector into embryonic brains at E13.5 and cell localization was analyzed at E15.5 by anti-GFP immunostaining. GFP and DAPI signals are shown in green and blue, respectively. VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bar: 100 μm. E Quantification of GFP+ cell positions in (D) (n = 3).


The NICD plasmid was electroporated with the Ttyh1 knockdown vector ( shTtyh1 ) into embryonic brains at E13 . 5 and cell localization was analyzed at E14 . 5 by anti - GFP immunostaining . Scale bar : 100 μm . G Quantification of GFP + cell positions in ( F ) ( n = 3 ) .

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The NICD plasmid was electroporated with the Ttyh1 knockdown vector (shTtyh1) into embryonic brains at E13.5 and cell localization was analyzed at E14.5 by anti-GFP immunostaining. Scale bar: 100 μm. G Quantification of GFP+ cell positions in (F) (n = 3).


Effects of Ttyh1 expression on the generation of NICD at 2 days posttransduction of E14 . 5 neural progenitor cells were analyzed by Western blotting using an anti - NICD antibody . Quantification of band

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Effects of Ttyh1 expression on the generation of NICD at 2 days posttransduction of E14.5 neural progenitor cells were analyzed by Western blotting using an anti-NICD antibody. Quantification of band intensities


Effects of Ttyh1 knockdown ( C ) on the generation of NICD at 2 days posttransduction of E14 . 5 neural progenitor cells were analyzed by Western blotting using an anti - NICD antibody . ( B , D ) Quantification of band

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Effects of Ttyh1 knockdown (C) on the generation of NICD at 2 days posttransduction of E14.5 neural progenitor cells were analyzed by Western blotting using an anti-NICD antibody. (B, D) Quantification of band intensities


Ttyh1 - transduced E14 . 5 neural progenitors were incubated with DAPT for 2 days , then cells were harvested and lysates were used for luciferase assay ( n = 3 ) .

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Ttyh1-transduced E14.5 neural progenitors were incubated with DAPT for 2 days, then cells were harvested and lysates were used for luciferase assay (n = 3).


Effects of Ttyh1 - knockdown on γ - secretase activity was also assessed using E14 . 5 neural progenitor

Effects of Ttyh1-knockdown on γ-secretase activity was also assessed using E14.5 neural progenitor cells


Ttyh1 - transduced E14 . 5 neural progenitors were incubated for 2 days in the presence or absence of DAPT , and samples were prepared for Western blotting using anti - NICD

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Ttyh1-transduced E14.5 neural progenitors were incubated for 2 days in the presence or absence of DAPT, and samples were prepared for Western blotting using anti-NICD antibody


Ttyh1 - transduced E14 . 5 neural progenitors were incubated for 2 days in the presence or absence of DAPT , and samples were prepared for qPCR for Notch target genes ( I )

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Ttyh1-transduced E14.5 neural progenitors were incubated for 2 days in the presence or absence of DAPT, and samples were prepared for qPCR for Notch target genes (I)


The indicated plasmids were electroporated into E13 . 5 brains , and brain samples were harvested at E15 . 5 for anti - GFP immunolabeling . GFP and DAPI signals are shown in green and blue , respectively . Quantification of GFP + cell localization in ( J ) dnPS1 , dominant negative presenilin 1 ; VZ , ventricular zone ; SVZ , subventricular zone ; IZ , intermediate zone ; CP , cortical plate . Scale bar : 100 μm .

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The indicated plasmids were electroporated into E13.5 brains, and brain samples were harvested at E15.5 for anti-GFP immunolabeling. GFP and DAPI signals are shown in green and blue, respectively. Quantification of GFP+ cell localization in (J) dnPS1, dominant negative presenilin 1; VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bar: 100 μm.


Effects of the ion selectivity - perturbing R371Q mutation in Ttyh1 E14 . 5 neural progenitor cells were transduced with retroviral vectors expressing each indicated gene , and 2 days posttransduction , cells were harvested for qPCR analyses of Notch target mRNAs ( n = 3 ) .

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Effects of the ion selectivity-perturbing R371Q mutation in Ttyh1 E14.5 neural progenitor cells were transduced with retroviral vectors expressing each indicated gene, and 2 days posttransduction, cells were harvested for qPCR analyses of Notch target mRNAs (n = 3).


Effects of the ion selectivity - perturbing R371Q mutation in other Ttyh family members on Notch - dependent transcription . E14 . 5 neural progenitor cells were transduced with retroviral vectors expressing each indicated gene , and 2 days posttransduction , cells were harvested for qPCR analyses of Notch target mRNAs ( n = 3 ) .

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Effects of the ion selectivity-perturbing R371Q mutation in other Ttyh family members on Notch-dependent transcription. E14.5 neural progenitor cells were transduced with retroviral vectors expressing each indicated gene, and 2 days posttransduction, cells were harvested for qPCR analyses of Notch target mRNAs (n = 3).


Immunolabeling of E15 . 5 brain sections electroporated in utero with indicated plasmids at E13 . 5 using anti - GFP and Alexa Fluor 488 - conjugated secondary antibodies . GFP immunofluorescence ( green ) was merged with DAPI - counterstained images ( blue ) . ( D ) Quantification of GFP + cell localization in ( C ) ( n = 3 ) . VZ , ventricular zone ; SVZ , subventricular zone ; IZ , intermediate zone ; CP , cortical plate . Scale bar : 100 μm .

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Immunolabeling of E15.5 brain sections electroporated in utero with indicated plasmids at E13.5 using anti-GFP and Alexa Fluor 488-conjugated secondary antibodies. GFP immunofluorescence (green) was merged with DAPI-counterstained images (blue). (D) Quantification of GFP+ cell localization in (C) (n = 3). VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bar: 100 μm.


series of Ttyh1 mutants with deletions in the C - terminal cytoplasmic tail region , and ( F ) their effects on cell distribution assessed by intraventricular in utero electroporation at E13 . 5 followed by immunolabeling at E15 . 5 using anti - GFP antibody . Scale bar : 100 μm .

series of Ttyh1 mutants with deletions in the C-terminal cytoplasmic tail region , and (F) their effects on cell distribution assessed by intraventricular in utero electroporation at E13.5 followed by immunolabeling at E15.5 using anti-GFP antibody. Scale bar: 100 μm.


Representative Western blots showing coimmunoprecipitation ( coIP ) of HA - tagged Rer1 with Myc - tagged Ttyh1 using the HEK293T cell line . WCL , whole - cell lysates .

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Representative Western blots showing coimmunoprecipitation (coIP) of HA-tagged Rer1 with Myc-tagged Ttyh1 using the HEK293T cell line. WCL, whole-cell lysates.


Venus fluorescent protein - based bimolecular fluorescence complementation analysis of Ttyh1 and Rer1 interaction ( green ) in living COS - 7 cells by fluorescence microscopy . Ttyh1 and Rer1 were tagged with N - and C - terminal fragments of the Venus fluorescent protein ( VN and VC ) , respectively . DsRed2 - ER was cotransfected to label the endoplasmic reticulum ( ER , red ) . Scale bar : 25 μm .

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Venus fluorescent protein-based bimolecular fluorescence complementation analysis of Ttyh1 and Rer1 interaction (green) in living COS-7 cells by fluorescence microscopy. Ttyh1 and Rer1 were tagged with N- and C-terminal fragments of the Venus fluorescent protein (VN and VC), respectively. DsRed2-ER was cotransfected to label the endoplasmic reticulum (ER, red). Scale bar: 25 μm.


CoIP assays were performed with anti - Ttyh1 and anti - Rer1 antibodies to show endogenous interaction between Ttyh1 and Rer1 in U373MG cells .

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CoIP assays were performed with anti-Ttyh1 and anti-Rer1 antibodies to show endogenous interaction between Ttyh1 and Rer1 in U373MG cells.


Western blot analyses of endogenous Rer1 proteins were performed at 2 days posttransduction using E14 . 5 primary neural progenitor cells transduced with retroviral vectors expressing ( D ) Ttyh1 Quantification of relative band

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Western blot analyses of endogenous Rer1 proteins were performed at 2 days posttransduction using E14.5 primary neural progenitor cells transduced with retroviral vectors expressing (D) Ttyh1 Quantification of relative band intensities


D - G Western blot analyses of endogenous Rer1 proteins were performed at 2 days posttransduction using E14 . 5 primary neural progenitor cells transduced with retroviral vectors expressing shTtyh1 . Quantification of relative band

D-G Western blot analyses of endogenous Rer1 proteins were performed at 2 days posttransduction using E14.5 primary neural progenitor cells transduced with retroviral vectors expressing shTtyh1. Quantification of relative band intensities


Cell extracts from primary neural progenitor cells transduced with Ttyh1 retroviruses in the presence of DMSO vehicle or a proteasome inhibitor lactacystin ( 10 μM ) , were immunoblotted with an antibody to Rer1 .

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Cell extracts from primary neural progenitor cells transduced with Ttyh1 retroviruses in the presence of DMSO vehicle or a proteasome inhibitor lactacystin (10 μM), were immunoblotted with an antibody to Rer1.


HEK293T cells were transfected with Ttyh1 - Myc and Rer1 - HA expression plasmids , and after 24 h , cells were treated with cycloheximide ( CHX ) ( 100 μg / ml ) and then harvested at the indicated time intervals . Cell lysates were analyzed by Western blotting using anti - HA antibody . ( J ) Quantification of band intensities in ( I ) ( n = 3 ) .

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HEK293T cells were transfected with Ttyh1-Myc and Rer1-HA expression plasmids, and after 24 h, cells were treated with cycloheximide (CHX) (100 μg/ml) and then harvested at the indicated time intervals. Cell lysates were analyzed by Western blotting using anti-HA antibody. (J) Quantification of band intensities in (I) (n = 3).


γ - secretase - dependent luciferase activity was measured using primary neural progenitor cells transduced with Ttyh1 with or without Rer1 retroviral vectors at 2 days posttransduction ( n = 3 ) .

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γ-secretase-dependent luciferase activity was measured using primary neural progenitor cells transduced with Ttyh1 with or without Rer1 retroviral vectors at 2 days posttransduction (n = 3).


Effects of Rer1 expression on Ttyh1 activity in vivo were examined by in utero electroporation into E13 . 5 brains and subsequent GFP immunofluorescence of E15 . 5 brain sections . GFP and DAPI signals are shown in green and blue , respectively . ( M ) Distribution of GFP + cells in each brain layer in ( L ) was quantified ( n = 3 ) . VZ , ventricular zone ; SVZ , subventricular zone ; IZ , intermediate zone ; CP , cortical plate . Scale bar : 100 μm .

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Effects of Rer1 expression on Ttyh1 activity in vivo were examined by in utero electroporation into E13.5 brains and subsequent GFP immunofluorescence of E15.5 brain sections. GFP and DAPI signals are shown in green and blue, respectively. (M) Distribution of GFP+ cells in each brain layer in (L) was quantified (n = 3). VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bar: 100 μm.


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

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


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

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


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

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


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

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


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

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


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

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


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

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


( A ) Upper panel , left : MCF - 7 / CD44 + 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 .

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


( B ) Upper panel : Mammosphere formation was performed by seeding 20 , 000 MDA - MB - 231 / EpCAM + 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 .

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


( C ) In vivo limiting dilution assays . miR - 10 - OE or control 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 .

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


( D ) Stemness 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 .

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


( B ) MCF - 7 , SKBR - 3 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 .

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


( C ) PTEN 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 .

(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 .

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


( E ) PTEN 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 .

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


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

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


( G ) miR - 10b overexpression does not increase mammosphere formation in PTEN - depleted 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 .

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


( H ) T47 - D 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 .

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


( I ) PTEN overexpression compensates for miR - 10b overexpression . Left panel : MDA - 468 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 ) .

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


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

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


( B ) Upper panel . Cultures were serum - starved for 18 h 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 .

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(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 .

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


( D ) miR - 10b overexpression does not induce additional changes in 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 .

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


Tissue 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

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Tissue 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 test


Manual 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 )

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Manual 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

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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 indi


Two - tailed Spearman correlation test between PAR and γH2AX ( % positive with a median intensity cut - off ) . Exact p values are indicated when

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Two-tailed Spearman correlation test between PAR and γH2AX (% positive with a median intensity cut-off). Exact p values are indicated when available


Top , 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

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Top, 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 changed


Top , 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

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Top, 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 changed


Genes 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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Genes 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


Indicated 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 =

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Indicated 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=0


ChIP - qPCR after C4 - 2 cells were treated as depicted in Figure 2 . Data are depicted as mean + / - standard deviation of three independent biological

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ChIP-qPCR after C4-2 cells were treated as depicted in Figure 2. Data are depicted as mean +/- standard deviation of three independent biological experiments


Athymic 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

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Athymic 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 tumors


Prostatectomy 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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Prostatectomy 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


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


Left : 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

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Left: 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 transcriptome


Left : 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

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Left: 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 each


Prostatectomy 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

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Prostatectomy 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 test


C4 - 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

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C4-2 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 promoter


C4 - 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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C4-2 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


Expression 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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Expression 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


Indicated 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 .

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Indicated 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=0.2781


Indicated 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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Indicated 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


( 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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(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).


( 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 .

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(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 .

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(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 .

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(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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(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).


( 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 .

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(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 .

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

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 .

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

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 .

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(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 .

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(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.