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. 2011 Sep;179(3):1257–1264. doi: 10.1016/j.ajpath.2011.05.041

Figure 3.

Figure 3

Down-regulation of GLTSCR2 sensitizes cells to DNA damage. A–B: Control (SK-shScr) and GLTSCR2 knockdown (SK-shGLT, 1 and 2) cells were exposed to the indicated doses of UV (A) or treated with the indicated concentrations of NCS (B). Clonogenic assays were then performed, as described in Materials and Methods, with the number of surviving colonies shown here. All data are from the three independent experiments are presented as mean ± SD (P < 0.01). C: Indicated cells were treated as in panel A and co-immunostainined with anti-phospho-histone H3 (pH3) and anti-active-caspase-3 (caspase-3a) antibodies. Data represent the mean numbers of cells that were positive for both pH3 and caspase-3a as determined by three independent experiments. One hundred cells were counted per sample. D–E: Excessive, persistent DNA damage in GLTSCR2-knockdown SK-shGLT cells. Repair of DSBs was detected in SK-shScr and SK-shGLT cells at the indicated time points both before and after exposure to UV (D) or NCS (E). The histograms display relative comet tail length quantification, as measured by comet tail movement and normalized to control cells (SK-shScr). Assays were performed in quadruplicate, and all data are presented as mean ± SD. The lower panels denote representative examples of single cells that were gel electrophoresed under alkaline conditions and subsequently stained with SYBR Green to demonstrate fast-migrating damaged DNA in the comet tails. Scale bar = 10 μm.