Figure 6.

DNA damage response is suppressed in HIF-α-expressing cells. (a) RCC4 and RCC4/VHL cells were treated with 2 μM CPT, 100 μM etoposide (ETO) or 400 nM of the mTORC1/2 kinase inhibitor pp242 for 24 h. Whole-cell lysates were assayed by western blot for phosphorylated p53 (S15), CHK1 (S317, S345), γH2AX (S139), total p53 and CHK1 proteins. Actin was used as a loading control. (b) The 786-O and 786-O/VHL cells were transfected with siRNA to ATR or nonsilencing control (NSC) for 24 h before addition of 2 μM CPT for a further 24 h. mRNA expression of ET-1 was assessed by real-time quantitative PCR relative to GAPDH. Mean±S.E. of duplicate values of one representative experiment is shown. (c) RCC4/VHL cells were treated with 10 μM ATM inhibitor (ATMi), 400 nM pp242 or vehicle control (DMSO) in the absence (−) or presence (+) of 2 μM CPT for 24 h. Whole-cell lysates were assayed by western blot for phosphorylated p53 (S15), and p53 proteins. Short and long exposure times are shown for P-S15-p53. Actin was used as a loading control. (d) RCC4/VHL and 786-O/VHL cells were preincubated with 10 μM ATMi for 1 h before addition of 2 μM CPT for a further 24 h and mRNA expression of ET-1 was assessed by real-time quantitative PCR relative to GAPDH. Mean±S.E. of duplicate values of one representative experiment is shown