PMC:4608092 / 22011-26447
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4608092","sourcedb":"PMC","sourceid":"4608092","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4608092","text":"Induction of RNF213 in HeLa Cells and HUVECs\nWe investigated the induction of RNF213 in HeLa cells and HUVECs after treatment of various angiogenic factors (TGF-β, IL-1β, VEGF, and PDGF-BB) and antiangiogenic factors (IFN-α, IFN-β, and IFN-γ). After 24 hours of treatment, none of the factors, except for IFN-γ, changed RNF213 protein levels in HeLa cells (Figure3). However, IFN-γ increased RNF213 protein levels in a dose- and time-dependent manner (Figure3A and 3B) with corresponding increases in mRNA levels (Figure3C and 3D). We then investigated the effects of treatment with the same factors on HUVECs at the 24-hour time point. Whereas TGF-β, IL-1β, VEGF, and PDGF-BB did not change RNF213 protein levels (Figure4A), IFN-β and IFN-γ, but not IFN-α, increased protein levels in a dose-dependent manner (Figure5). IFN-β and IFN-γ also induced RNF213 in a time-dependent manner (Figure4B and 4C). We further examined mRNA levels of RNF213 after treatment with IFN-β and IFN-γ. Both factors increased mRNA levels, but IFN-β increased mRNA levels at a lower dose (0.1 ng/mL) and an earlier time point (3 hours) than IFN-γ (Figure4D through 4G).\nFigure 3 Screening the effects of angiogenic factors and antiangiogenic factors on RNF213 protein and mRNA expression in HeLa cells. A and B, HeLa cells were treated with various concentrations of angiogenic factors and antiangiogenic factors for 24 hours (A), or in the presence of 10 ng/mL of IFN-γ for the indicated times (B), and levels of RNF213 protein expression were examined by western blotting analysis using β-tubulin as a loading control. C and D, HeLa cells were treated with various concentrations of IFN-γ for 24 hours (C) or in the presence of 10 ng/mL of IFN-γ for the indicated times (D). RNA samples were analyzed by qPCR using PPIA as an internal control. Fold induction by IFN-γ was compared with activities of 0 ng/mL of IFN-γ. A column represents a mean of 3 independent experiments. Bars indicate SD. *P\u003c0.05, by Student t test compared with IFN-γ 0 ng/mL. IFN-γ indicates interferon γ; IL, interleukin; PDGF-BB, platelet-derived growth factor; qPCR, quantitative polymerase chain reaction; TGF-β, transforming growth factor β; VEGF, vascular endothelial growth factor.\nFigure 4 Screening effects of angiogenic factors and antiangiogenic factors on RNF213 protein and mRNA expression in HUVECs. A through C, HUVECs were treated with various concentrations of angiogenic factors for 24 hours (A), or in the presence of 10 ng/mL of IFN-β (B) or IFN-γ (C) for the indicated times, and levels of RNF213 protein expression were examined by western blotting analysis using β-tubulin as a loading control. D through G, HUVECs were treated with various concentrations of IFN-β (D) or IFN-γ (F) for 24 hours or in the presence of 10 ng/mL of IFN-β (E) or IFN-γ (G) for the indicated times. RNA samples were analyzed by qPCR using PPIA as an internal control. Fold induction by IFN-β or IFN-γ was compared with activities of 0 ng/mL of IFN-β or IFN-γ. A column represents a mean of 3 independent experiments. Bars indicate SD. *P\u003c0.05, by Student t test compared with 0 ng/mL IFN-β or IFN-γ. HUVECs indicates human umbilical vein endothelial cells; IFN, interferon; IL, interleukin; PDGF-BB, platelet-derived growth factor; qPCR, quantitative polymerase chain reaction; TGF-β, transforming growth factor β; VEGF, vascular endothelial growth factor.\nFigure 5 Effects of IFNs on RNF213 protein expression in HUVECs. HUVECs were treated with various concentrations of IFNs for 24 hours, and RNF213 protein expression was examined by western blotting analysis using β-tubulin as a loading control. Representative western blotting findings are shown in upper panel. A column represents a mean of 3 independent experiments (lower panel). Bars indicate SD. *P\u003c0.05, by Student t test compared with 0 ng/mL IFNs. HUVECs indicates human umbilical vein endothelial cells; IFNs, interferons. These findings indicated that IFN-β increased RNF213 expression levels in HUVECs in a vascular EC-specific manner, whereas IFN-γ did not. The other factors had no effect on expression levels of RNF213. Upregulation of RNF213 protein was preceded by an increase in RNF213 mRNA levels, which suggested that IFN-β increased RNF213 expression at the transcriptional level. Because IFN-β induced RNF213 in HUVECs, we chose IFN-β for further characterization because of its tissue specificity.","divisions":[{"label":"Title","span":{"begin":0,"end":44}},{"label":"Figure caption","span":{"begin":1149,"end":2245}},{"label":"Figure caption","span":{"begin":2244,"end":3415}},{"label":"Figure caption","span":{"begin":3414,"end":3948}}],"tracks":[]}