Fig. 8: PAARH physically bound to HIF-1α, recruited HIF-1α to VEGF promoter, and activated VEGF transcription.

a RIP assays followed by qRT-PCR to detect RNAs bound to HIF-1α. b RNA pull-down experiments using RNA end-labeled with desthiobiotin, followed by western blot to detect proteins bound to PAARH. c SNU-398 cells with PAARH overexpression or control were incubated under hypoxia or normoxia for 24 h, followed by ChIP with anti-HIF-1α antibody or nonrelated IgG. Precipitated DNAs were measured by qRT-PCR for VEGF promoter. d SNU-398 cells with PAARH silencing or control were incubated under hypoxia or normoxia for 24 h, followed by ChIP with anti-HIF-1α antibody or nonrelated IgG. Precipitated DNAs were measured by qRT-PCR for VEGF promoter. e Luciferase activity in SNU-398 cells with PAARH overexpression or control after transfection of luciferase reporters containing VEGF promoter or nothing. Results are shown as the relative ratio of firefly luciferase activity to renilla luciferase activity. f Luciferase activity in SNU-398 cells with PAARH silencing or control after transfection of luciferase reporters containing VEGF promoter or nothing. Results are shown as the relative ratio of firefly luciferase activity to renilla luciferase activity. g VEGF expression in SNU-398 cells with PAARH overexpression and concurrent HIF-1α depletion under hypoxia for 12 h was measured by qRT-PCR. h The number of endothelial tubes formed by HUVEC treated with conditioned medium from SNU-398 cells with PAARH overexpression and concurrent VEGF depletion under hypoxia for 24 h. Scale bars, 200 µm. i Schematic model of the roles of PAARH in regulating HCC cellular malignancy and angiogenesis. Results are shown as mean ± SD based on three independent experiments. **p < 0.01, ***p < 0.001, ns not significant, by Student’s t test (a, c, e) or one-way ANOVA followed by Dunnett’s multiple comparisons test (d, f–h).