Figure 4.

Poly(I:C) increases the production of active VEGF through HIF-1α. (A and B) vegf mRNA and protein levels of PC3 cells were evaluated after 5 µg/ml poly(I:C) stimulation for the indicated time points using qRT-PCR (A) and ELISA (B), respectively. (C and D) Formation of capillary-like structures. Representative phase-contrast microphotographs of capillary-like structures (C) and quantitative evaluation of their morphogenesis in HUVEC cultures (D). HUVECs were exposed to supernatants of PC3 cells untreated (ctr) or treated with poly(I:C) for 48 hours in the presence or absence of VEGF neutralizing antibodies (anti-VEGF Ab). HUVEC exposed to serum-free medium (DMEM) with or without poly(I:C) for 48 hours were used as negative controls. (E) Cells were transiently transfected with a vector encoding an RNA interfering hif-1α (int. hif-1α) or with a vector encoding a scramble RNA sequence (int. scramble), then treated with 5 µg/ml poly(I:C) for 6 hours and analyzed for total HIF-1α protein level using Western blot. β-Actin was used as a control for equal amounts of protein loaded. (F) Transfected cells were treated with poly(I:C) for 24 hours, and vegf mRNA expression was evaluated by qRT-PCR. The histograms in A, B, D, and F represent the mean of triplicate samples from three independent experiments with SD as error bars, *P ≤. 05, **P ≤. 01. Data shown in panel E are typical of three separate experiments with similar results.