Enhancement of SRF Transcriptional Activity by HDAC6 Inhibitor and siRNA Silencing
(A, B) Luciferase assay for serum response factor (SRF) transcriptional activity. (C) Western blots showing efficient histone deacetylase 6 (HDAC6) knockdown. For luciferase assay, MOVAS cells in 10% fetal bovine serum (FBS) culture were transfected with the E1350 construct for 6 h. Transfected cells were selected with hygromycin B, recovered in Dulbecco’s modified Eagle’s medium (DMEM) containing high glucose and 10% FBS for 24 h, and then seeded in 24-well plates at a density of 20,000 cells/well. After overnight starvation in DMEM (high glucose, 0.5% FBS), cells were (A) treated with vehicle or 5 μmol/l tubastatin A for 24 h or (B) transfected with scrambled small interfering ribonucleic acid (siRNA) or HDAC6-specific siRNA for 24 h or 48 h, followed by lysis in Bright-Glo for luciferase assay. For each bar graph, at least 3 independent experiments were performed; mean ± SEM; *p < 0.05 compared with (A) nontransfected or (B) scrambled control, #p < 0.05 compared between vehicle and tubastatin A, analyzed with 1-sample Student’s t-test.