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. 2020 Apr 10;295(20):7060–7074. doi: 10.1074/jbc.RA119.012131

Figure 4.

Figure 4.

ZFHX3 is required for transactivation of the hypoxia-responsive VEGFA in HepG2 cells. A and B, knockdown of ZFHX3 reduced hypoxia-induced VEGFA expression, as analyzed by both real-time PCR (A) and ELISA (B), and experiments were performed in duplicate for each group. C and D, similarly, in Huh-7 cells, knockdown of ZFHX3 reduced hypoxia-induced (C) or DFO-induced (D) VEGFA expression, as analyzed by real-time PCR. E, hypoxia-induced HRE promoter luciferase activity was reduced by the knockdown of ZFHX3 and increased by ectopic expression of ZFHX3 in HepG2 cells. F, ZFHX3 was required for hypoxia to induce VEGFA expression in HepG2 cells under hypoxia, as measured by real-time PCR. G, knockdown of HIF1A dramatically reduced the binding of ZFHX3 to VEGFA promoter in HepG2 cells under hypoxia. Western blotting (lower panel) confirmed the knockdown effect. H and I, knockdown of ZFHX3 dramatically reduced and ectopic expression of ZFHX3 increased the binding of HIF1A to the promoter of VEGFA in HepG2 cells under hypoxia. IgG was used as the isotype control. Western blotting (lower panel) confirmed the knockdown effect. Data are shown as means ± S.D. The statistical analysis for both luciferase assay and real-time PCR was based on three independent experiments (n = 3), and the value for each group in an experiment was the average of triplicates. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant.