Fig 1: 53BP1 and RAP80 altered foci accumulation in FA pathway-deficient cells. (A) Representative images of RAP80 (red) and 53BP1 (green) foci in nuclei (DAPI stained, blue) of FANCC-mutated (PD331 FANCC−/−) and -corrected (PD331corr) cells 24 h after MMC exposure (1 μg/ml/1 h). White line: 2 μm. (B) Representative images of RIF1 (red) and 53BP1 (green) foci in nuclei (DAPI stained, blue) of FANCC-mutated (PD331 FANCC−/−) and -corrected (PD331corr) cells 24 hours after MMC exposure (1 μg/ml/1 h). White line: 2 μm. (C) 53BP1 and RAP80 foci quantificationin untreatyed cells (0 h) and at different time points (1, 6 and 24 h) following MMC exposure (1 μg/ml/1 h). Histograms represent the mean of three independent experiments. Error bars indicate S.D. The data were analyzed by a Student's t-test; ** indicates P < 0.01. (D) Size (arbitrary unit, a.u.) of 53BP1 foci in untreated conditions and 24 h after MMC exposure (1 μg/ml/1 h) in FANCC-mutated (PD331 FANCC−/−) and -corrected (PD331corr) cells. The cell size was determined using the ImageJ software. The values on the histogram represent the mean of three independent experiments; at least 50 cells were scored each time. Error bars indicate S.D. Data were analyzed by a Student's t-test; *** indicates P < 0.001. (E) Western blot showing the efficiency of the siRNA against 53BP1 and RAP80, as observed 72 h after transfection, in corrected (PD331 corr) and FA-C (PD331 FANCC−/−) cells. Vinculin was used as a loading control. siCtrl (control) indicates cells transfected with an untargeted siRNA. (F) Representative images of RAP80 (red) and 53BP1 (green) foci in nuclei (DAPI stained, blue) of FANCC-corrected cells (PD331 corr) after depletion of 53BP1 or RAP80 by siRNA transfection. Cells were treated with MMC (1 μg/ml/1 h) and analyzed 24 h later. White line: 2 μm. siCtrl (control) indicates cells transfected with an untargeted siRNA. (G) Quantification of RAP80 foci-positive cells after transfection with an untargeted siRNA (siCtrl, control), si53BP1 or siMDC1 in FANCC-mutated (PD331 FANCC−/−) and -corrected (PD331corr). Cells presenting more than 5 foci were considered positive. Data represent the mean of two independent experiments with similar results. (H) Left. Representative images of BRCA1/RAP80 interaction dots detected with the Proximity Ligation Assay in nuclei (DAPI stained, blue) in FANCC-mutated (PD331 FANCC−/−) and -corrected (PD331corr) cells under untreated conditions or 24 h following MMC exposure (1 μg/ml/1 h). White line: 8 μm. Right. BRCA1/RAP80 interaction dots quantification 24 h after MMC exposure (1 μg/ml/1 h). Histograms represent the pooled data from three independent experiments. Quantification was conducted using ImageJ software. (I) Left. Representative images of BRCA1/CtIP (left) interaction dots detected with the Proximity Ligation Assay in nuclei (DAPI stained, blue) in FANCC-mutated (PD331 FANCC−/−) and -corrected (PD331corr) cells under untreated conditions or 24 hours following MMC exposure (1 μg/ml/1 h). White line: 8 μm. Right. BRCA1/CtIP interaction dots quantification 24 h after MMC exposure (1 μg/ml/1 h). Histograms represent the pooled data from three independent experiments. Quantification was conducted using ImageJ software. (J) Representative images of FANCD2 foci (red), RAP80 (red) or 53BP1 (green) in PD20 corr (FANCD2-corrected cells), PD20 (FANCD2−/- cells) and PD20 FANCD2 K561R (PD20 cells expressing a K561R, non-ubiquitinable FANCD2) cells. White line: 2 μm. Histograms represent the number of RAP80 (red) and 53BP1 (green) foci in the three different cell lines in the left. Data represent the mean of two independent experiments with similar results.
Fig 2: NHEJ pathway inhibition rescue HR and cell survival in FA cells. (A) Representative images of pDNA-PKcs (red) and MRE11 (green) foci in nuclei (DAPI stained, blue) of FANCC-corrected (PD331 corr) or FANCC-mutated (PD331 FANCC−/−) cells in which 53BP1 was depleted by siRNA White line: 2 μm. (B) Histogram presents the frequency of MRE11-positive FANCC−/− cells 48 h after 53BP1 downregulation by siRNA transfection. The presented data are the mean of three independent experiments; error bars indicate S.D. *** indicates P < 0.001 using a Student's t-test. (C) Representative images of pDNA-PKcs (red), 53BP1 (green), RAP80 (red) or MRE11 (green) foci in nuclei (DAPI stained, blue) of FANCC-deficient cells (PD331 FANC−/−) treated with DMSO or with a DNA-PK specific inhibitor. The cells were treated with DNA-PK inhibitor (DNA-PKi 10 μm) for 2 h before MMC exposure (200 ng/ml). White line: 2 μm. (D and E) Clonogenic survival of FANCC-proficient (D, PD331 corr) or -mutated (E, PD331 FANCC−/−) cells after 53BP1 depletion by siRNA and/or DNA-PK inhibition. The cells were treated with MMC at the indicated doses. The presented data are the mean of three independent experiments; error bars indicate S.D. * indicates P < 0.05 using a Student's t-test.
Fig 3: SOX9 directly binds the DDR gene promoters and induces their expression.A Venn diagram illustrates the intersections between core DDR genes (n = 276) and SOX9 target genes (n = 263) identified by ChIP-Seq. B Cells were treated with or without olaparib (SKOV3, 10 µM; UWB1.289, 2 µM) for 48 h. qPCR analysis of ChIP samples from experiments performed in SKOV3 and UWB1.289 cells using the anti-SOX9 antibody or IgG. C qPCR was performed to detect the mRNA levels of PAXIP1, SMARCA4, UIMC1, SLX4, and TDP2 in cells stably transfected with pLKO.1 (shNC), SOX9 shRNA 1 (shSOX9-1), or SOX9 shRNA 2 (shSOX9-2). Cells were treated with olaparib (SKOV3, 10 µM; UWB1.289, 2 µM) for 48 h before harvest. D qPCR was performed to detect the mRNA levels in cells stably transfected with pCMV or pCMV SOX9. Cells were treated with olaparib (SKOV3, 10 µM; UWB1.289, 2 µM) for 48 h before harvest. E Western blot was performed to detect the protein levels of SMARCA4, UIMC1, SLX4, and SOX9 in cells stably transfected with pLKO.1 (shNC), SOX9 shRNA 1 (shSOX9-1), SOX9 shRNA 2 (shSOX9-2), pCMV, or pCMV SOX9. Cells were treated with olaparib (SKOV3, 10 µM; UWB1.289, 2 µM) for 48 h before harvest. F Quantification of the protein levels in (E). G Cells transfected with pLKO.1 (shNC), USP28 shRNA 1 (shUSP28-1), or USP28 shRNA 2 (shUSP28-2) were challenged with olaparib for 48 h, and the protein levels of SMARCA4, UIMC1, SLX4, USP28, and SOX9 were detected using western blot. H Cells transfected with pCMV, pCMV USP28, and SOX9 shRNA 1 (shSOX9-1) were challenged with olaparib for 48 h, and the protein levels were detected using western blot. I Cells were treated with AZ1 (10 µM) and/or olaparib for 48 h. Western blot was used to detect protein levels. Quantification of protein levels in Fig. 6G–I was shown in Supplementary Fig. S9B–D. (Data are presented as the mean ± SEM, *p < 0.05, **p < 0.01, n = 3).
Supplier Page from Abcam for Anti-RAP80 antibody [EPR5315]