Statistical Analysis
Results are presented as mean±SD. Number of samples are provided in the figure legends. Statistical tests on in vitro experiments were performed using the Student t test to detect the effect of treatments by comparing controls according to study designs shown below.
We first screened several angiogenic and antiangiogenic cytokines to determine whether treatment with different doses of these cytokines induced RNF213 in cultured cells. IFN-β and IFN-γ were found to induce RNF213. We then statistically compared RNF213 levels in IFN-β- or IFN-γ-treated cells with untreated cells. The mechanism of RNF213 induction with IFN-β was examined in cells transfected with RNF213 WT or mutated promoter luciferase plasmid. To test the effect of a STATx mutation, luciferase activity in cells transfected with STATx mutation promoter was compared with cells transfected with RNF213 WT promoter. Effects of treatment of IFN-β on angiogenesis were statistically evaluated by tube formation and migration assay using HUVECs and iPSECs. In experiments of target protein depletion (p-STAT1, STAT1, or RNF213) using corresponding siRNAs, we compared target protein levels in cells treated with target siRNA with cells treated with control siRNA. We also tested the effect of STAT1 and RNF213 siRNA on angiogenesis.
To evaluate the in vitro effect of various RNF213 mutations on angiogenesis, the angiogenic function of HUVECs transfected with RNF213 mutants were compared with HUVECs transfected with a control vector. In addition, ATPase activities of RNF213 mutants were compared to RNF213 WT.
For in vivo animal studies, both nonparametric methods (Kruskal–Wallis 1-way ANOVA followed by Mann–Whitney U test) and a parametric method (2-way ANOVA method) were conducted to detect effects of treatment (hypoxia), genotype, and interaction on cerebral angiogenesis. Cerebral angiogenesis was evaluated by the increase in the numbers of cerebral microvessels/mm2.
Values of P<0.05 were considered statistically significant. Statistical analyses were performed using SAS software (version 9.4; SAS Institute Inc., Cary, NC).