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. 2011 Dec 15;104(1):42–55. doi: 10.1093/jnci/djr509

Figure 2.

Figure 2

DNA damage and DNA damage responses after treatment with TMZ and/or G47Δ in GSCs in vitro. A) γH2AX induction in GBM8 cells (MGMT negative). Cells were mock treated or treated with TMZ (3 μM) for 36 hours and then mock infected or infected with G47Δ (MOI = 1). Twenty-four hours later, cells were fixed and processed for immunocytochemistry (DAPI, blue; anti-γH2AX, green; anti-HSV, red). B) γH2AX induction in BT74 cells (MGMT positive). Cells were mock treated or treated with TMZ (200 μM) + BG (50 μM) for 36 hours and then mock infected or infected with G47Δ (MOI = 1) in the presence of BG (50 μM). Twenty-four hours later, cells were processed as in (A). Scale bar = 100 μm in (A) and (B). C) The ratio of γH2AX-positive nuclei/total was determined in three randomly selected fields with GBM8 (upper panel) and BT74 with BG (50μM) (lower panel). Combi = TMZ + G47Δ (GBM8); TMZ + BG + G47Δ (BT74). Groups are significantly different in GBM8 and BT74 (P < .001, one-way ANOVA). Asterisks indicate statistically significant differences between indicated pairs (P < .05, Bonferroni Multiple Comparison Test). Error bars represent 95% confidence intervals. D) GSCs were treated as in (A), harvested 24 hours later, and processed for western blotting. Left panels: GBM4 (TMZ 20 μM), Middle panels: GBM8 (TMZ 3 μM), Right panels: BT74 (TMZ 200 μM, BG 50 μM). M: mock, T = TMZ, G = G47Δ, B: BG. E) DSBs induced by TMZ (+BG), G47Δ, or the combination, were assessed by neutral comet assay and plotted as individual tail moments, with the dark horizontal bars representing the means and 95% confidence intervals. Groups are significantly different in GBM4, GBM8, and BT74 (P < .001, one-way ANOVA). Asterisks indicate statistically significant differences between indicated pairs (P < .001, except for GBM4 Mock vs TMZ, P = .012, unpaired t test, two-sided). Experiments were performed in triplicate. ANOVA = analysis of variance; ATM = ataxia telangiectasia mutated; ATR = ataxia telangiectasia and Rad3-related; ATRIP = ATR interacting protein; BG = O6-benzylguanine; DAPI = 4′, 6-diamidino-2-phenylindole; DNA-PKcs = DNA-dependent protein kinase catalytic subunit; DSB = DNA double-strand break; GSC = glioblastoma stem cell; HSV = herpes simplex virus; MGMT = O6-methylguanine-DNA-methyltransferase; MOI = multiplicity of infection; p-ATM = phosphorylated ATM (Ser1981); p-ATR = phosphorylated ATR (Ser428); TMZ = temozolomide.