Fig 4.

PLD1 activity is required for IL-1β-induced expression of angiogenic factors via binding of HIF-1α to the promoters of its target genes. (A) RAFLS were pretreated with VU0155069 or transfected with two kinds of siRNAs specific to PLD1, followed by stimulation with IL-1β for 24 h. Secretion of VEGF and IL-8 was quantified by ELISA. (B and C) RAFLS were transfected with siRNA specific to PLD1, pretreated with or without VU0155069, and stimulated with IL-1β for 36 h. Cell lysates were immunoblotted with the indicated Abs. The relative levels of the indicated proteins were quantitated by densitometer analysis. The data shown are representative of four independent experiments. (D) RAFLS were transfected with HRE-Luc, pretreated with VU0155069, and stimulated with IL-1β for 36 h, and then luciferase activity was determined. (E) RAFLS were pretreated with VU0155069 or cycloheximide (CHX, 5 μM) for 30 min, treated with IL-1β for 36 h, and then treated with MG132 (20 μM) for 6 h. The HIF-1α protein level was determined by Western blotting. Relative HIF-1α protein levels were quantitated by densitometer analysis. The data shown are representative of four independent experiments. (F and G) RAFLS were pretreated with VU0155069 for 30 min and treated with IL-1β for 12 h, and a ChIP assay was performed with preimmune IgG, anti-HIF-1α, and anti-NF-κB Abs, followed by qPCR. *, P < 0.01; **, P < 0.05. The data presented are the means ± SDs of four independent experiments.