Figure 3

Immunocytochemical analysis of irradiation-induced repair foci. A. Evaluation of immunocytochemical evaluation of γH2AX, 53P1 and MDC1 foci after irradiation with either 1.5 Gy (upper panel) or 6 Gy (bottom panel) using conventional fluorescence microscopy. HCC1395 cells were compared with MCF10A cells as a wildtype control and with HCC1937 as a BRCA1 mutant control. Under these assay conditions, the numbers of γH2AX and MDC1 foci appeared significantly reduced in p.R215W mutant cells (HCC1395) at 30 min after irradiation, with 53BP1 foci being slightly reduced. No such reduction was observed in HCC1937 cells. HCC1937 exhibited increased residual levels of foci at 24 hours and 48 hours after irradiation. Data represented as mean & SEM, *p < 0.05, **p < 0.01, ***p < 0.001, n = 4. B, C. Radiation-induced MDC1 foci (B), γH2AX and 53BP1 foci (C). Representative images are shown for the immunocytochemical analysis of MDC1 (green, Figure B), γH2AX (green, Figure C) and 53BP1 (red, Figure C) foci at 30 min after irradiation with 6 Gy with confocal microscopy revealing discrete and more diffuse foci formation in HCC1395 compared to wildtype cells (MCF10A). Scale bar = 5 μm. D. Enlarged area of single nuclei from B, C for quantitative evaluation. Note the smaller area and fuzzy appearance of foci in the HCC1395 cell line. E. Analysis of foci area in HCC1395 and wildtype cells (MCF10A) for γH2AX, 53P1 and MDC1. For each, areas of 60–90 foci were analysed. Data are represented as mean & SEM. *p < 0.05.