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Fig. 6. siRNA knockdown of cyclophilin B enhances ER stress-induced cell death. (A) Knockdown efficiency of CypB-siRNA was determined by western blotting. ER chaperon proteins such as Grp94, Bip and PDI were detected as a reference to monitor the effect of CypB-siRNA on other ER chaperone proteins. (B) MTT assay. Relative cell viability of each cell line is shown based on that of the con-siRNA transfection. The data are means ± s.d. obtained from five independent experiments. *P<0.05 versus Tg-treated con-siRNA; #P<0.05 versus Tm-treated con-siRNA. (C) Western blotting with apoptosis marker antibodies: caspase 3 (cleaved form) and Bax. TUNEL assay performed after Tg treatment for 24 hours in con-siRNA and CypB-siRNA transfection. Green arrows indicate TUNEL-positive cells. Numbers under each band are representative of at least five different experiments and are expressed as the means ± s.d. *P<0.05 versus Tg-treated con-siRNA transfection. (D) The defensive role of CypB is partly caspase dependent. PcDNA-, CypB/wt- and CypB-siRNA-transfected cells were cultured with 50 µM z-VAD-fmk prior to Tg treatment. The percentage of viable cells was determined using the MTT assay. The data are expressed as the means ± s.d. obtained from five independent experiments. *P<0.05 versus Tm-treated pcDNA transfectant; #P<0.05 versus Tm-treated CypB-siRNA transfectant.
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