Figure 2.

TNF-α induced ANRIL expression is mediated by NF-κB activation. (A) Schematic representation of ANRIL promoter region and the predicted NF-κB binding site. TSS means transcriptional start site. (B and C) NF-κB p65 (B) and ANRIL (C) RNA levels in HUVECs transfected with scramble or 2 sets of NF-κB p65 siRNAs (sip65–1 and sip65–2, 24h) upon TNF-α treatment (25 ng/mL, 12h). (*p < 0.05, **p < 0.01, ***p < 0.005). (D) CHIP analysis showing the increased binding of p65 to ANRIL promoter in HUVECs upon TNF-α treatment (25 ng/mL, 6h). Binding of p65 to IL8 promoter was used as a positive control, and binding of p65 to GAPDH promoter was a negative control. (*p < 0.05, n.s. means non-significant). (E) ANRIL levels in HUVECs treated with TNF-α (25 ng/mL), IL-1β (10 ng/mL), LPS (1 μg/mL), IFN-γ (100 ng/mL), EGF (10 ng/mL), PDGF-BB (10 ng/mL), VEGF₁₆₅ (50 ng/mL) and insulin (100 nmol/L) for 24 hours. (*p < 0.05, ***p < 0.005 vs. control). (F) Representative immunoblot showing the p65 phosphorylation (ser536) in HUVECs treated with factors mentioned in (E) for 30 min. β-tubulin was used as an internal control. (G) Histogram showing pooled quantification data of p65 phosphorylation from immunoblot experiments in (F) (*p < 0.05 vs. control). All results are presented as mean ± SEM from at least 3 independent biological experiments.