Role of RNF213 in Antiangiogenic Activity of IFN-β
First, we evaluated effects of treatment with IFN-β on HUVEC proliferation, and found that IFN-β did not increase proliferation (Figure7). Angiogenic activity was then evaluated by tube formation and migration assays. We confirmed that IFN-β lowered tube formation and inhibited migration (Figures8 and 9) without affecting proliferation rates (Figure7). Because IFN-β induced RNF213, we hypothesized that antiangiogenic activity of IFN-β is mediated by RNF213. To test this hypothesis, we first depleted STAT1 by siRNA. Antiangiogenic activity of IFN-β, except for branching, was normalized by depletion of STAT1 and phosphorylated STAT1 by siRNA transfection (Figure10A and 10B). Notably, siRNA also downregulated RNF213 (Figure10A). This downregulation was likely mediated by activation of the promoter. We then depleted RNF213 protein levels by siRNA transfection. Depletion of RNF213 restored tube formation and migration (Figures1 and 2). These rescue experiments by siRNA transfection demonstrated that RNF213 was involved in antiangiogenic activity of IFN-β in ECs.
Figure 7  Effects of IFN-β on HUVEC proliferation as evaluated by trypan blue dye exclusion tests. At 1 day after HUVEC seeding, 0 or 10 ng/mL of IFN-β was added. Data with bars represent mean±SD (n=3). No significant difference (P<0.05) between 0 ng/mL of IFN-β (blue line) and 10 ng/mL of IFN-β (red line) was observed at each time point according to Student t test. HUVEC indicates human umbilical vein endothelial cell; IFN, interferon.
Figure 8  Antiangiogenic activity of IFN-β in HUVECs. A, Tube formation assays in HUVECs after 20 hours of culture with IFN-β on Matrigel. A concentration of 0 ng/mL of IFN-β was used as a positive control (100%). Scale bars indicate 100 μm. Representative images are shown in left panel. The tubal areas were quantified by imaging analysis (right panel). Data with bars represent mean±SD (n=3). *P<0.05 according to Student t test compared with 0 ng/mL of IFN-β. B, Migration assays for HUVECs after treatment with IFN-β (1 ng/mL). A concentration of 0 ng/mL of IFN-β was used as a control. Scale bars indicate 100 μm. Representative images are shown in left panel. Re-endothelialized areas were quantified by imaging analysis (right panel). Data with bars represent mean±SD (n=3). *P<0.05 according to Student t test compared with 0 ng/mL of IFN-β. HUVECs indicates human umbilical vein endothelial cells; IFN, interferon.
Figure 9  Tube formation by HUVECs after 20 hours of culture on Matrigel with IFN-β. IFN-β (0 ng/mL) served as the positive control (100%). Total tube length and number of branches were quantified by imaging analysis. Data with bars represent mean±SD (n=3). *P<0.05 according to Student t test compared with 0 ng/mL of IFN-β. HUVECs indicates human umbilical vein endothelial cells; IFN, interferon.
Figure 10  Effects of STAT1 depletion on RNF213 expression and antiangiogenic activities of IFN-β in HUVECs. A, Western blot analyses of RNF213 and STAT1 protein expressions and STAT1 protein phosphorylation (at Ser727; p-STAT1) in HUVECs treated with IFN-β for 24 hours after control or STAT1 siRNA transfection. β-tubulin served as the loading control. Representative western blot experiments are shown in the upper panel. A column with a bar (lower panel) represents mean±SD (n=3). *P<0.05 according to Student t test compared with 0 ng/mL of IFN-β with the same siRNA treatment. #P<0.05, cells treated with 10 ng/mL of IFN-β were compared between STAT1 siRNA treatment and no siRNA treatment using Student t test. †P<0.05, STAT1 siRNA treatment was compared with control siRNA at 10 ng/mL of IFN-β using Student t test. B, Tube formation assays for HUVECs cultured with IFN-β on Matrigel after control or STAT1 siRNA transfection. Treatment with 0 ng/mL of IFN-β after control siRNA transfection was evaluated as a positive control (100%). Scale bars indicate 100 μm. Representative images are shown in upper panel. The tube area, total tube length, and number of branches were quantified by imaging analysis (lower panel). A column with a bar represents mean±SD (n=3). *P<0.05, according to Student t test comparing 0 ng/mL of IFN-β within the same siRNA treatment paradigm. HUVECs indicates human umbilical vein endothelial cells; IFN, interferon; p-STAT1, phosphorylated signal transducer and activator of transduction 1.
Figure 11  Effects of RNF213 depletion on antiangiogenic activity of IFN-β in HUVECs. A, Western blot analysis of RNF213 protein expression in HUVECs treated with IFN-β for 24 hours after control or RNF213 siRNA transfection. β-tubulin served as the loading control. Representative western blotting results are shown in left panel. A column with a bar (right panel) represents mean±SD (n=3). *P<0.05, according to Student t test comparing 0 ng/mL of IFN-β within the same siRNA treatment paradigm. #P<0.05, cells exposed to 10 ng/mL of IFN-β were compared between RNF213 siRNA treatment and no treatment using Student t test. †P<0.05, using Student t test comparing control siRNA at 10 ng/mL of IFN-β. B, Tube formation assays for HUVECs cultured with IFN-β on Matrigel after control or RNF213 siRNA transfection. Treatment with 0 ng/mL of IFN-β after control siRNA transfection was used as a positive control (100%). Scale bars indicate 100 μm. Representative images are shown in left panel. Tube area was quantified by imaging analysis (right panel). A column with a bar represents mean±SD (n=3). *P<0.05, according to Student t test comparing 0 ng/mL of IFN-β within the same siRNA treatment paradigm. C, Migration assays for HUVECs treated with IFN-β (1 ng/mL) after control or RNF213 siRNA transfection. Treatment with 0 ng/mL of IFN-β after control siRNA transfection served as the control. Scale bars indicate 100 μm. Representative images are shown in left panel. Re-endothelialized areas were quantified by imaging analysis (right panel). A column with a bar represents mean±SD (n=3). *P<0.05, according to Student t test comparing 0 ng/mL of IFN-β within the same siRNA treatment paradigm. HUVECs indicates human umbilical vein endothelial cells; IFN, interferon.
Figure 12  Tube formation assays for HUVECs after 20 hours of culture on Matrigel with IFN-β after control or RNF213 siRNA transfection. Treatment with 0 ng/mL of IFN-β after control siRNA transfection served as the positive control (100%). Total tube length and number of branches were quantified by imaging analysis. A column with a bar represents means±SD (n=3). *P<0.05, by Student t test compared with 0 ng/mL of IFN-β within the same siRNA treatment. HUVECs indicates human umbilical vein endothelial cells; IFN, interferon; iPSECs, induced pluripotent stem cell-derived vascular endothelial cells.

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