Fig. 5. Downregulation of eIF3k protects cells from apoptosis. (A) Generation of eIF3k siRNA stable transfectants. HeLa cells were co-transfected with plasmid expressing eIF3k-specific siRNA or control siRNA and the plasmid pEGFP/Neo^r (carrying both GFP and the neomycin-resistant gene) at a ratio of 4:1. Cells were selected with G418. For transfectants carrying the control vector, all G418-resistant clones were pooled. eIF3k-siRNA transfectants were first tested for the expression of eIF3k and seven clones displaying a reduced level of eIF3k were pooled. The two pools of stable transfectants were lysed and subjected to western blot with antibodies to eIF3k and tubulin. (B-D) HeLa cells stably expressing eIF3k siRNA or control siRNA as described in A were assayed for apoptosis by flow-cytometry analysis of cells with fragmented DNA (sub-G1 DNA). The percentage of cells with fragmented DNA is plotted. In B and D, cells were treated with 10 ng/ml TNF together with 2.5 ng/ml Actinomycin D (B) or 1 µM staurosporine (STS, D), respectively, for various durations. In C, cells were irradiated with UV at 0.015 J/cm² and then cultured for various durations as indicated. (E) Clonogenic survival of UV-irradiated cells carrying eIF3k siRNA or control siRNA. HeLa cells as in A were irradiated with UV at the indicated doses. Cells were cultured for 8 days and then stained by crystal violet. Colony numbers were quantitated by ImageJ software. The percentage of clonogenic survival was determined by the ratio of colony numbers derived from the treated population to those from the untreated population. (F) Expression of the siRNA-resistant eIF3k (eIF3k^R) construct reverses the apoptosis-modulating effect of eIF3k siRNA. HeLa cells transfected with constructs as indicated were assayed for apoptosis induced by UV or TNF (bottom panel), or the level of eIF3k (upper panel). For B-F, data shown are mean ± s.d. from three independent experiments. ^*P<0.05; ^**P<0.005.