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. 2021 Feb 15;131(4):e135296. doi: 10.1172/JCI135296

Figure 7. HIF-2α upregulates Sema3G expression upon hypoxia in ECs.

Figure 7

(A and B) Sema3G and Vegfa mRNA levels in bEnd.3 cells exposed to normoxia (21% O2) or hypoxia (1% O2) for the indicated times. Data were normalized to gene expression in cells upon normoxia (n = 3 independent experiments). (C) Immunoblot analysis of Sema3G, HIF-1α, and HIF-2α protein in bEnd.3 cells exposed to hypoxia (1% O2) for the indicated times. (D and E) RT-qPCR analysis of Sema3G mRNA in bEnd.3 cells, which were transfected with siHIF-1α (D), siHIF-2α (E), or siControl for 48 hours and then exposed to hypoxia (1% O2) or normoxia (21% O2) for an additional 12 hours (n = 3 independent experiments). (F) Schematic diagram depicting the mouse Sema3G promoter with the presence of hypoxia response element (HRE) sequences. HRE sequences from the JASPAR database. ChIP-qPCR primers of the indicated HRE regions are shown. (G) ChIP-qPCR assays were performed with the antibodies against HIFs or IgG as control in bEnd.3 cells exposed to 1% O2 for 12 h (n = 3 independent experiments). (H) Diagrammatic representation of mutated HRE (mHRE) introduced into the mouse Sema3G promoter to test HREs in regulating Sema3G transcription. (I) Luciferase reporter assay for Sema3G promoter activity in HEK293 cells following transfection of different mHRE vectors (n = 3 independent experiments). Error bars represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; 2-tailed Student’s t tests (A, B, D, E, G), 1-way ANOVA with Dunnett’s multiple comparisons test (I). TSS, transcription start site.