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. 2018 Nov 9;294(1):28–37. doi: 10.1074/jbc.RA118.005392

Figure 3.

Figure 3.

Analysis of interplay between TRPC5, NFATc3, and ANGPT1 in ECs. A, top panel, detection of differentially regulated angiogenic proteins with an angiogenesis antibody array in WT and TRPC5−/− mouse serum samples. Duplicate spots: 1, Angiopoietin-1; 2, DPPIV; 3, Endostatin/Collagen XVIII; 4, IGFBP-1; 5, IGFBP-2; 6, IGFBP-3; 7, Leptin; 8, MMP-3; 9, MMP-9; 10, NOV; 11, Osteopontin; 12, PDGF-AA; 13, PDGF-AB/PDGF-BB; 14, Pentraxin-3; 15, Platelet Factor 4; 16, SDF-1; 17, Serpin E1; 18, Serpin F1. Bottom panel, quantitation of relative protein expression by comparing the mean pixel density of each factor in the TRPC5−/− group with that of each factor in the WT group. B, top, Western blotting of angiopoietin-1 (ANGPT1) in WT and TRPC5−/− primary MMECs with β-actin as a loading control. The image is representative of three independent experiments. Bottom, quantitation of ANGPT1 band intensity relative to β-actin. Values represent means ± S.E. of band intensities from three independent experiments. *, p < 0.05 versus WT, unpaired Mann–Whitney test. C, NFATc3 stimulates the transcriptional activity of ANGPT1 in HEK293 cells co-transfected with NFATc3 and a luciferase reporter plasmid carrying the 5′ flanking the 2012-bp sequence of ANGPT1. An empty vector was transfected as a control. The horizontal axis indicates relative luciferase activity. n = 5. ***, p < 0.001 versus vector, unpaired t test with Welch's correction. D and E, Western blots (left panels) and quantitation (right panels) of NFATc3 (n = 6) and ANGPT1 (n = 3) expression in primary MMECs (D) and HMECs (E) transfected with NFATc3 siRNA or scrambled siRNA. The band intensities were normalized to β-actin and quantified using ImageJ. ***, p < 0.001 versus control (Ctl), Student's unpaired two-tailed t test. *, p < 0.05 versus Ctl, unpaired Mann–Whitney test. F and G, representative images of the tube formation in primary MMECs (F) and HMECs (G) treated with TRPC5 siRNA, NFATc3 siRNA, or control scrambled siRNA. The data are means ± S.E. n = 5 to 8. F, **, p < 0.01; ***, p < 0.001 versus Ctl; Kruskal–Wallis test and Dunn's multiple comparisons test. G, ***, p < 0.001 versus Ctl, one-way ANOVA and Dunnett's multiple comparisons test. Scale bars = 50 μm. A–E, representative images are shown, and the values are means ± S.E.