Figure 2.

Identification of a target gene responsible for induction of apoptosis by genotoxic stress. (A) The identified shRNA (No. 20) suppresses TAF1 gene. 293T cells were transfected with pEGFP-C1 vector or the shRNA clone No. 20. Total RNA was subjected to RT–PCR analysis using primer sets for TAF1 or GAPDH. 293T cells were also transfected with scramble siRNA or siRNA targeting TAF1. Cell lysates were subjected to immunoblot analysis with anti-TAF1 or antitubulin. (B) TAF1 gene-specific shRNA or siRNA inhibits induction of apoptosis by genotoxic stress. 293T cells transfected with pUC19 vector or the shRNA clone No. 20 were left untreated (open bar) or treated (closed bar) with H₂O₂ for 24 h. The percentage of apoptotic cells was determined by TUNEL assays. The data represent the mean ± SD from three independent experiments, each performed in triplicate. An asterisk indicates P < 0.05. (C) 293T cells transfected with scramble siRNA, TAF1 siRNA or caspase-3 siRNA were left untreated (open bar) or treated (closed bar) with H₂O₂ for 24 h. The percentage of apoptotic cells was analyzed as described earlier. The data represent the means ± SD from three independent experiments, each performed in triplicate. Two asterisks indicate P < 0.01. Cell lysates were subjected to immunoblot analysis with anti-caspase-3 or antitubulin. (D) Ectopic expression of TAF1 induces apoptosis. U2OS cells were transfected with pEGFP-C1 vector or GFP-TAF1 and untreated (open bar) or treated (closed bar) with H₂O₂ for 24 h. The percentage of apoptotic cells was determined by TUNEL assays. The data indicate the means ± SD from three independent experiments, each performed in triplicate. Two asterisks indicate P < 0.01. Cell lysates were subjected to immunoblot analysis with anti-GFP or antitubulin.