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Fig. 7. eIF3k regulates apoptosis in a K8/K18-dependent manner. (A) Generation of eIF3k-siRNA stable transfectants. EBNA-SW13 cells were co-transfected with a plasmid expressing eIF3k siRNA or control siRNA and a plasmid carrying the puromycin-resistant gene, and then selected with puromycin. Pools of stable transfectants were established as described in Fig. 5 and then used to analyze the expression of eIF3k by western blot. (B-D) Pools of stable transfectants as described in A were treated with 10 ng/ml TNF together with 2.5 ng/ml Actinomycin D (B), or 1 µM staurosporine (D) for various durations, as indicated. Alternatively, cells were irradiated with UV at 0.015 J/cm² and then cultured for the indicated durations (C). (E) EBNA-SW13 cells carrying eIF3k siRNA or control siRNA as shown in A were co-transfected with pDR2-K8 and pDR2-K18. The transfectants were selected by hygromycin and then lysed for western blot analysis with antibodies as indicated. Lysate of the parental EBNA-SW13 cells was included to reveal the lack of K8/K18 expression in this cell line. (F,G) SW13-K8/K18 cells carrying eIF3k siRNA or control siRNA as shown in E were treated with 10 ng/ml TNF together with 2.5 ng/ml Actinomycin D (F) or 1 µM staurosporine (G) for various durations as indicated. Apoptotic cells were analyzed as in Fig. 5. Data shown are mean ± s.d. from three independent experiments. ^*P<0.05; ^**P<0.005.

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