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. 2015 Sep 24;30(1):441–456. doi: 10.1096/fj.15-278648

Figure 6.

Figure 6.

Supplementation with H2S rescues transcription of specific genes and endothelial phenotype. A) Effect of GSH and NaHS supplementation on the relative luciferase activity of −116/+268 VEGFR-2 promoter in scrambled or CBS siRNA-treated HUVECs after 48 h transfection with siRNA, −116/+268 VEGFR-2 promoter-firefly luciferase, and wild-type pRLTK-Renilla luciferase. Data represent means ± sd (n = 3), 2-tailed Student’s t test. *P ≤ 0.05, **P ≤ 0.01. B) Effect of increasing dose of NaHS supplementation for 24 h on the cell viability of HUVECs determined by MTT assay. Data represent means ± sd (n = 3), 1-way ANOVA; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, #P > 0.05 (not significant). C) Immunoblot analysis of VEGFR-2, NRP-1, and Sp1 protein levels in scrambled and CBS siRNA-treated HUVECs after treatment with various doses of NaHS for 24 h. NaHS treatment was initiated 24 h posttransfection and continued for 24 h after which after immunoblotting was performed. D) Immunoblot analysis of VEGFR-2, NRP-1, and Sp1 protein levels in scrambled and CBS siRNA-treated HUVECs after treatment with various doses of reduced GSH. NaHS treatment was initiated 24 h posttransfection and continued for 24 h after which immunoblotting was performed. E) Rescue of VEGF A165-induced HUVEC proliferation in CBS-silenced HUVECs with various doses of GSH, NaHS or GYY4137. Statistics analysis was performed between VEGF165-treated CBS-silenced HUVECs with metabolite (GSH/NaHS/GYY4137) supplemented VEGF165-treated CBS-silenced HUVECs. Data represent means ± sd (n = 3), 1-way ANOVA; *P ≤ 0.05, **P ≤ 0.01. F) Rescue of VEGF A165-induced HUVEC chemotaxis in CBS-silenced HUVECs with or without treatment with 2 mM doses of GSH or NaHS. GSH or NaHS treatment was initiated 24 h posttransfection and treatment carried out for another 24 h before performing the chemotaxis assay. i) Scrambled control siRNA + VEGF A165 (10 ng/ml). ii) CBS siRNA + VEGF A165 (10 ng/ml). iii) CBS siRNA + GSH (2 mM) + VEGF A165 (10 ng/ml). iv) CBS siRNA + VEGF A165 (10 ng/ml). Data represent means ± sd (n = 3), 1-way ANOVA. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, #P > 0.05.