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. Author manuscript; available in PMC: 2010 Jan 13.
Published in final edited form as: Mol Cell. 2008 Feb 29;29(4):488–498. doi: 10.1016/j.molcel.2007.12.029

Figure 2. Overexpression of exogenous Bcl2 down-regulates Ku activity in association with decreased DSB repair and increased genetic instability.

Figure 2

(A) WT Bcl2/pCIneo DNA construct was stably transfected into H1299 human lung cancer cells. Expression of Bcl2 was determined by Western blot. (B) DNA binding activity of Ku 70 or Ku 86 was measured using a Ku70/86 DNA Repair Kit. Error bars represent ± S.D. (C–E) H1299 cells overexpressing WT Bcl2 or vector control were exposed to 1 Gy of ionizing radiation (IR). Cells were harvested at various time points. DSBs were determined by PFGE or analysis of γ-H2AX by immunostaining and Western blot. The number of γ-H2AX foci per cell was determined on a cell to cell basis by the quantitative analysis of at least 30 randomly chosen cells as described (Balajee et al., 2004). The percentage of γ-H2AX foci positive cells was determined by analyzing 100 randomly chosen cells as described (Chowdhury et al., 2005). Levels of γ-H2AX protein from Western blot were quantified by densitometry. Error bars represent ± S.D. (F) The percentage of abnormal metaphases from Bcl2 overexpressing cells or vector-only control cells was quantified by T-FISH analysis. At least 30 metaphases per culture were analyzed. Error bars represent ± S.D. (G) Frequency of cytogenetic abnormality per metaphase in Bcl2 overexpressing cells or vector-only control cells. Each experiment was repeated three times and error bars represent ± S.D.