Figure 4

Caspase-dependent apoptosis in poly(I:C)-transfected RCC cell lines. (a) Two RCC lines were cultured or transfected with poly(I:C) (SKRC-1, 500 ng/ml; SKRC-44, 50 ng/ml). After 24 h, cleaved forms of a panel of caspases and PARP were evaluated by immunoblotting. β-actin was used as a control. Similar results were obtained from two independent experiments. (b) RCC lines were cultured or transfected with poly(I:C) (SKRC-1 and SKRC-44, 500 ng/ml) in the presence of the indicated caspase inhibitors (20 μM). The percentage of Annexin V⁺ cells was determined by flow cytometry. Results are presented as the means ± standard deviations of three wells. **p < 0.01 compared with poly(I:C) transfection alone. Similar results were obtained from two independent experiments. PanCi, pan-caspase inhibitor; C8i, caspase-8 inhibitor; C9i, caspase-9 inhibitor; C2i, caspase-2 inhibitor. (c) Caspase-2 protein expression was examined by immunoblotting 3 days after transfection with control siRNA or caspase-2 siRNA. α-tubulin was used as a control. (d) RCC cells, which were pre-transfected with the indicated siRNAs 3 days prior, were transfected additionally with poly(I:C) (SKRC-1, 1,000 ng/ml; SKRC-44, 500 ng/ml). The percentage of Annexin V⁺ cells was determined by flow cytometry after 24 h. Results are presented as the means ± standard deviations of three wells. **p < 0.01. Similar results were obtained from two independent experiments. (e) After poly(I:C) transfection (SKRC-1, 1,000 ng/ml; SKRC-44, 500 ng/ml), the expression of caspases was evaluated by immunoblotting. β-tubulin was used as a control. Similar results were obtained from two independent experiments. pIC-TF, poly(I:C) transfection.