Figure 5.

RB Family Protein Loss Impairs DNA Damage Clearance

(A) DSB clearance in cells with compromised RB1 function. Wild-type, RB1^−/−, or RB1/RBL1/RBL2-null (TKOs) mouse embryonic fibroblasts (MEFs) were exposed to IR of 5 Gy. Prevalence of damage at the indicated times was detected by immunofluorescence staining for γH2AX (green); nuclei were visualized with Hoechst 33258 (blue). Raw images recorded using a high-content imaging platform are shown. Scale bar represents 10 μm.

(B and C) Automated quantitative assessment of damage clearance. γH2AX staining intensity (iSig) was determined for all cells from 30 independent eye fields. (B) Integrated H2AX signal distribution for cells from one representative experiment. The dotted line shows the gating position for cells with above baseline anti-γH2AX fluorescence. DNA content analysis for the same samples shown in Figure S5. (C) Percentage of cells with residual damage determined by gating (dotted line) for cells with above baseline anti-γH2AX fluorescence at each time point. Error bars represent ±SD for n = 3 biological replicates. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001 using a paired Student’s t test comparing RB1^−/− or TKOs to wild-type cells.

(D) Immunodetection documenting RB protein family loss in MEFs. Lysate from MEFs used for (A)–(C) were probed using antibodies as indicated.