Fig. 5.

Base modifications in the VEGF HRE that occur after perinuclear clustering mitochondria are required for HIF-1α binding and VEGF mRNA expression. (A) Top: Western analyses of HIF-1α and the nuclear marker lamin A/C in PAECs cultured for 3 hours under normoxia (NORM) or hypoxia (HYP) in the presence of nocodazole (Noc) or after transfection with dynein-specific siRNA (siDYN). Representative of four experiments. Bottom: Quantification of HIF-1α abundance normalized to lamin A/C calculated as a percentage of the normoxic control. n = 4 separate culture dishes per experimental group. *P < 0.05, increased from normoxia. (B) Western blot analysis of HIF-1α associating with a 65-mer oligonucleotide model of the VEGF HRE (DNA affinity precipitation analysis). Oligonucleotide-associated HIF-1α was derived from nuclear extracts isolated from normoxic and hypoxic control PAECs or PAECs treated with nocodazole or transfected with dynein-specific siRNA. Data are representative of three separate experiments. (C) ChIP analysis of VEGF HRE sequences immunoprecipitating with HIF-1α recovered from PAECs incubated under normoxia or hypoxia in the presence of nocodazole. (D) ChIP assays for VEGF HRE sequences immunoprecipitating with HIF-1α from PAECs transfected with dynein-specific (siDYN) or scrambled siRNA (Scram). (E) Quantitative RT-PCR analysis of VEGF mRNA expression by PAECs in the presence of nocodazole. (F) Quantitative RT-PCR analysis of VEGF mRNA expression in PAECs transfected with dynein-specific or scrambled siRNA. n = 4 to 6 separate culture dishes per experimental group. *P < 0.05, increased from normoxia. **P < 0.05, different from normoxia and hypoxia alone.