Fig 1: BMI1/RAD51 axis inhibition enhanced replication stress of ALDHbr bCSCs.A, B Bar plots representing the proportion of γH2AX foci in ALDHneg (A) and ALDHbr (B) SUM159 cells sorted according to their cell-cycle phases, after 24 h/72 h of treatments with RAD51i or DMSO (CTRL). C–F Quantification of replication-stress markers in ALDHbr and ALDHneg SUM159 cells treated with RAD51i or DMSO (CTRL). C Representative images of active replication clusters (green staining). Scale bar: 5 μm. D Bee-swarm plots representing the proportion of replication clusters per nucleus in each SUM159 cell subpopulation. E Representative images of combed DNA molecules. CldU (green staining) and IdU (red staining) were detected using specific antibodies and DNA is counterstained with DAPI (blue staining). Scale bar: 10 μm. F Bee-swarm plots representing the distribution of fork speed in each cell subpopulation. G–J Bar plots representing the proportion of RAD51-positive (G, H) or γH2AX-positive cells (I, J) in ALDHneg (G, I) and ALDHbr (H, J) SUM159 cells sorted according to their cell-cycle phases, after treatment with BMI1i or DMSO (CTRL). K Representative examples of flowchart for the ALDEFLUOR staining following BMI1i or RAD51i treatment in SUM159 cells. DEAB is an ALDH inhibitor used as negative control. L, N Bar plot representing the proportion of ALDHbr cells following BMI1i (L) or RAD51i (N) treatment compared with the untreated condition (CTRL). M, O Bar plot representing tumorsphere-forming efficiency (SFE) for SUM159 cells under BMI1i (M) or RAD51i (O) treatment compared with untreated conditions (CTRL). Statistical test used is Student’s t-test. Data represent mean ± SD.
Fig 2: BMI1 nuclear location affects replicative stress response in breast cancer cells.A Representative images of BMI1 staining (green bodies). Nuclei are counterstained with DAPi (blue staining). BMI1 protein can accumulate in CAP bodies (left panel) or has uniform punctate distribution (PcG bodies, right panel). Scale bar: 5 μm. B Stacked bar plot representing the proportion of CAP and PcG bodies in ALDHbr and ALDHneg SUM159 cells sorted according to their cell-cycle phase (G0/G1, S, and G2/M). C, D Representative images (left panels) of BMI1 costaining (green foci) with γH2AX (red foci) for cells harboring CAP bodies (C) or PcG bodies (D). Nuclei are counterstained with DAPi (blue staining). The red lines correspond to the line scans. Pearson’s coefficient evaluated the amount of colocalization. Scale bar: 5 μm. On the right panels, line-scan profiles of the relative intensity of BMI1 and γH2AX fluorescent signals. E Bar plots representing the proportion of replicative ALDHbr and ALDHneg SUM159 cells with BMI1/γH2AX colocalization. F, G Representative images (left panels) of BMI1 costaining (green foci) with RAD51 (red foci) for cells harboring CAP bodies (F) or PcG bodies (G). Nuclei are counterstained with DAPi (blue staining). The red lines correspond to the line scans. Pearson’s coefficient evaluated the amount of colocalization. Scale bar: 5 μm. On the right panels, line-scan profiles of the relative intensity of BMI1 and RAD51 fluorescent signals. H Bar plots representing the proportion of replicative ALDHbr and ALDHneg SUM159 cells with BMI1/RAD51 colocalization. I Representative images of BMI1 staining (green bodies) in ALDHbr and ALDHneg SUM159 cells treated with 5-aza or untreated (CTRL). Nuclei are counterstained with DAPi (blue staining). J Stacked bar plot representing the proportion of CAP and PcG bodies in replicative ALDHbr and ALDHneg SUM159 cells treated with 5-aza or untreated. K Bar plots representing the proportion of γH2AX-positive cells for each cell subpopulation (replicative ALDHbr and ALDHneg SUM159 cells) following 5-aza treatment or in untreated conditions (CTRL). Statistical test used is Student’s t-test. Data represent mean ± SD.
Fig 3: ALDHbr bCSCs resist to cisplatin in a BMI1/RAD51-dependent manner.A Representative examples of flowchart for the ALDEFLUOR staining following cisplatin treatment in SUM159 cells. DEAB is an ALDH inhibitor used as negative control. B Bar plot representing the proportion of ALDHbr cells following cisplatin treatment compared with the untreated condition (CTRL). C Bar plot representing tumorsphere-forming efficiency (SFE) for SUM159 cells under cisplatin-treated and -untreated conditions. D Waterfall plot representing the range of change in ALDHbr bCSC proportion in PDXs treated with cisplatin. Positive changes represent enrichment in bCSCs and negative changes in reduction of bCSCs compared with the untreated conditions. A two-sided chi-square test evaluates the probability to predict bCSC response to cisplatin according to the presence of HR gene mutation in PDX. E Detection of labeled DNA–pt lesions (green staining) in ALDHbr and ALDHneg SUM159 cells using clickable cisplatin derivative APPO. Nuclei are counterstained with DAPI (blue staining). Scale bar: 5 μm. F Bar plot representing the proportion of ALDHbr and ALDHneg SUM159 cells with DNA-pt lesions at different time points following APPO treatment. G Bee swarm plots representing the distribution of forks speed in each cell subpopulations untreated or treated with cisplatin. H Kinetic curves tracing the proportion of RAD51-positive or γH2AX-positive cells following cisplatin treatment, in each cell subpopulations. I Stacked bar plot representing the proportion of CAP and PcG bodies in ALDHbr and ALDHneg SUM159 cells treated with 6 h/24 h of cisplatin compared with untreated condition (CTRL). J Bar plot representing the proportion of γH2AX-positive cells in ALDHbr and ALDHneg SUM159 cells treated with cisplatin alone or in combination with RAD51i or BMI1i. Statistical test used is Student’s t-test. Data represent mean ± SD.
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