Figure 5.

Overexpression of I.3 hif-1α in LNCaP cells determines poly(I:C)-induced VEGF production. (A) LNCaP cells were treated for the indicated times with 25 µg/ml poly(I:C), and Western blot analysis of HIF-1α was performed on nuclear extracts. Cells treated with 100 µM CoCl₂ for 3 hours were used as positive control for HIF-1α accumulation. β-Actin was used as a loading control. (B, C, and D) LNCaP cells were transiently transfected with a plasmid containing the I.3 isoform of HIF-1α (hif-1α I.3) or with a control plasmid (pcDNA3). Cells were then treated with 5 µg/ml poly(I:C) for 6 hours, and HIF-1α protein levels were analyzed using Western blot (B). Analysis of the I.1 and I.3 isoform of hif-1α mRNA was performed by using qRT-PCR (C). ELISA was used to analyze VEGF concentration in the CM of untransfected or transfected cells with the indicated plasmids and then treated or left untreated with poly(I:C) (D), the histogram represents the mean ± SEM of three independent experiments. **P ≤ .01. Data shown in panels A and B are typical experiments repeated three times with similar results. Histograms in panels C and D represent the mean of triplicate samples from three independent experiments with SD as error bars, *P ≤ .05, **P ≤ .01.