Fig. 3
Genomic instability in radiation repair protein 52 (RAD52)-depleted breast cancer susceptibility protein-1 (BRCA1)-deficient breast cancer cells. a Representation photomicrographs of immunofluorescence staining of DNA double strand break marker, phosphorylated histone 2A family member X (γ-H2AX), showed various levels of nuclear foci formation in MDA- MB-436 BRCA1-/- cells with or without endonuclease/exonuclease/phosphatase family domain-containing-1 (EEPD1) and/or RAD52 depletion. b Quantitative analysis of γ-H2Ax foci-positive (≥5 foci per cell) cell population in each condition. Cells were counterstained with 4',6-diamidino-2-phenylindole (DAPI). Each set of data was collected from five different × 40 fields from three distinct experiments. Mean + SEM is shown. Scale = 25 uM. c Western blot analysis of EEPD1, RAD52, phosphorylated ataxia-telangiectasia mutated kinase (ATM) and Rad3-related kinase (p-ATR), ATR (total), phosphorylated checkpoint kinase 1 (p-CHK1), CHK1 (total), p-ATM (phosphorylated), ATM (total), p-CHK2 (phosphorylated), CHK2 (total), phosphorylated p-replication protein A 32 kDa subunit (p-RPA32), RPA32 (total) and cyclophilin B (CypB) as a loading control in EEPD1and/or RAD52 depleted cells. d Cytogenetics showing an increase in chromosome breakage after RAD52 depletion in BRCA1-/- cells. Representative images of metaphases from each condition. Arrowhead indicates sites of chromosome breakage. e Quantitation of chromosomal breakage in each condition, with mean ± SEM shown (three distinct experiments with > 20 metaphases counted per experiment)