Normal RB1+/− cells have elevated γH2AX foci, mitotic errors, and aneuploidy. Normal RB1+/− cells have elevated γH2AX foci, mitotic errors, and aneuploidy. A, immunofluorescence microscopy for γH2AX in control and RB1+/− patient fibroblasts. Scale bars, 10 μm. B, quantitation of γH2AX foci in control and each patient fibroblast isolate. Foci were compared for patient fibroblasts against pooled control data (IMR90, BJ, and WI-38) using a χ2 test. Pooled control, n = 246; GM_01408, n = 79; GM_01123, n = 93; GM_06418, n = 71. C, Western blotting was used to detect phosphorylated RPA pS33 and total RPA32. Relative intensity of RPA pS33 is displayed. D, cells were transduced with H2B-GFP, and video microscopy was performed to capture phase contrast and GFP images over 15 hours. The left-most image shows the onset of prophase, and the middle image is of the metaphase plate just before the onset of anaphase (elapsed time since the onset of prophase is shown in yellow). The right-most image shows cells in telophase. Scale bars, 50 μm. E, summary of video microscopy data showing the number of divisions observed along with the number that contained anaphase bridges. Similarly, the number of cells observed to complete prophase and metaphase is shown along with the average length of these two phases. Each patient fibroblast is compared with pooled control data. #, P < 0.05, χ2 test; †, P < 0.05, t test. F–I, quantitation of genomic abnormalities in mesenchymal-derived cancer cell lines that are wild-type (n = 15), hemizygous (n = 10), or null (n = 12) for RB1. Means were compared using a t test. *, P < 0.05. NS, not significant. Courtney H. Coschi et al. Cancer Discovery 2014;4:840-853 ©2014 by American Association for Cancer Research