RAD52 inactivation is synthetically lethal with deficiencies in BRCA1 and PALB2 in addition to BRCA2 through RAD51-mediated homologous recombination - PubMed (original) (raw)

Figure 2

RAD51 foci formation in BRCA1-deficient cells is dependent on RAD52, but RAD52 foci formation is not dependent on BRCA1. (a) HCC1937 cells, complemented with a pcDNA3 expression vector containing wildtype BRCA1 or empty control, as previously reported, were transfected with the indicated siRNAs and immunoblotted with the indicated antibodies (anti-BRCA1: OP92, Calbiochem, La Jolla, CA, USA; anti-Rad52: SC-8350, Santa Cruz, Santa Cruz, CA, USA; anti-SMC1: A300-055A, Bethyl, Montgomery, TX, USA; anti-HDAC: #06-720, Upstate/Millipore, Billerica, MA, USA). Confocal images of IR-induced RAD51 foci in HCC1937 cells that were stably transfected with wildtype BRCA1 or a vector control. Cells were then transiently transfected with RAD52 siRNA or a control siRNA, then seeded in eight-chamber tissue culture slides (Fisher, Pittsburgh, PA, USA) and incubated overnight. Cells were fixed in 4% paraformaldehyde at room temperature for 15 min, followed by a block and permeabilization step with 10% BGS and 0.5% Triton-X for 1 h. Commercially available antibodies against RAD51 (Ab-1, Santa Cruz) were used for staining, as previously described. Images were obtained using a Carl Zeiss confocal laser scanning microscope (Carl Zeiss Microscopy, LLC, Thornwood, NY, USA) and processed using ImageJ and Adobe Photoshop software. For foci quantification 200 nuclei were counted, with cells forming > 5 foci scored as positive. The percentage of cells with RAD51 foci is shown (mean and s.e. from three independent experiments). Differences between HCC1937 cells stably transfected with an empty vector and then control siRNA compared with empty vector cells transfected with RAD52 siRNA are significant (_P_=0.0234), as determined by an unpaired _t_-test. (b) MCF7 cells were transfected with GFP-tagged RAD52 and a pool of cells were stably selected with G418 (Mediatech, Inc., Manassus, VA, USA) at 1 mg/ml concentration. These MCF7-RAD52-GFP cells were transfected with control or BRCA1 siRNA. After 10 Gy of IR, cells were fixed and stained with commercially available antibodies against BRCA1 (SC-6954, Santa Cruz) and phosphorylated Ser-139 H2AX (05-636, Millipore, Billerica, MA, USA). Confocal images of RAD52-GFP, BRCA1 and gamma-H2AX (gH2AX) foci were captured and scored. The percentage of cells with RAD52 and BRCA1 foci is shown (mean and s.e. from three independent experiments). Differences in BRCA1 foci between control and BRCA1 siRNAtransfected cells were significant (_P_=0.002), as determined by an upaired _t_-test.