Figure 3

DNA damage and p53 activation in poly(I:C)-transfected RCC cells. SKRC-1 and SKRC-44 cells were transfected with poly(I:C) (SKRC-1, 1,000 ng/ml; SKRC-44, 500 ng/ml) and cultured for 24 h in the presence or absence of NAC (10 mM). (a) The levels of phosphorylated γH2AX (Ser 139) in untreated controls (gray) and poly(I:C)-transfected cells (bold line) were examined by flow cytometry. Numbers represent the mean fluorescence intensities (MFI) of poly(I:C)-transfected cells. Similar results were obtained from three independent experiments. (b) The level of phosphorylated γH2AX (Ser 139) was examined by immunoblotting. NAC (10 mM) was added 6 h after poly(I:C) transfection. β-actin was used as a positive control. Similar results were obtained from two independent experiments. (c) Kinetic levels of total p53, phosphorylated p53 (Ser 15), NOXA, Puma, Bid, and tBid were examined by immunoblotting. β-actin was used as a control. Similar results were obtained from two independent experiments. (d) P53 protein expression was examined by immunoblotting 3 days after transfection with control siRNA or p53 siRNA. α-tubulin was used as a control. (e) RCC cells, which were pre-transfected with the indicated siRNAs 3 days prior, were transfected additionally with poly(I:C). The percentage of Annexin V⁺ cells were then determined by flow cytometry after 24 h. **p < 0.01. Similar results were obtained from two independent experiments. (f) Kinetic levels of total p53, NOXA and Puma were examined by immunoblotting. β-actin was used as a control. Similar results were obtained from two independent experiments. pIC-TF, poly(I:C) transfection.