Research Paper Volume 3, Issue 1 pp 33—43

Figure 3. HIPK2-induced p53 apoptotic activity is impaired by HIF-1α. (A) Luciferase assay in 293 cells co-transfected with Noxa-luc reporter and HIPK2-GFP (4 μg) expression vector alone or in combination with HIF-1α-Flag (8 μg). Results represent mean ± s.d. from three experiments. The expression of the ectopic proteins was assayed by immunoblot. (B) Lysates from RKO cells co-transfected with HIF-1α and HIPK2 expression vectors were assayed for RT-PCR of p53 target gene Noxa. GAPDH is a loading control (C) Luciferase assay in H1299 cells co-transfected with Noxa-luc reporter and low amount of wtp53 expression vector or of p53S46D mutant, in combination with HIPK2, HIF-1α or HIF-1αDN mutant. Results represent mean ± s.d. from three experiments. (D) Tunel assay of RKO cells where HIF-1α overexpression significantly reduced the HIPK2-induced apoptotic cell death. *P=0.001. (E) Luciferase assay in RKO cells stable transfected with p53AIP1-luc reporter where the adryamicin (ADR)-induced luciferase activity was inhibited by HIF-1α overexpression but not by HIF-1α dominat negative (DN) mutant. Results represent mean ± s.d. from three experiments. (F) Lysates from RKO cells treated as indicated were assayed for RT-PCR analyses of p53 apoptotic target Noxa and for HIPK2 expression. GAPDH is a loading control.