Figure 3.

Poly(I:C)-induced HIF-1α accumulation is TLR3-dependent. (A) PC3 cells stably transfected with a plasmid containing the dominant-negative form of TLR3 (PC3-TLR3-DN) or with a plasmid containing only the resistance to puromycin (PC3-PURO) were subjected to total DNA extraction, and the presence of the TLR3-DN plasmid was assessed using PCR with specific primers. Nontransfected PC3 cells and the plasmid used for the transfection (pZERO-hTLR3) were used as negative and positive controls, respectively. β-Actin was used as a control for equal amounts of DNA loaded. (B and C) Both stably transfected cell lines were treated for 6 hours with 5 µg/ml poly(I:C), and the increase of hif-1α mRNA and nuclear protein accumulation were evaluated by qRT-PCR (B) and Western blot analysis (C). β-Actin was used to normalize both qRT-PCR and Western blot. Data shown in panels A and C are typical of three separate experiments with similar results. The histogramin panel B represent the mean of triplicate samples from three independent experiments with SD as error bars, *P = .05.