Figure 1: Up-Regulation of SMC Marker Proteins by HDAC6 Inhibition Under PDGF-BB Stimulation

MOVAS cells were starved with 0.5% fetal bovine serum for overnight, and then pretreated with 5 μmol/l tubastatin A or 5 μmol/l RGFP966 or vehicle control (equal amounts of dimethylsulfoxide) for 2 h before the addition of 20 ng/ml of platelet-derived growth factor-BB (PDGF-BB). Data are quantified as fold changes versus control (normalized value as 1, condition specified later); mean ± SEM; n = 3 independent experiments; *p < 0.05 compared with control (one-sample Student’s t-test). (A) Western blot assay of vascular smooth muscle cell (SMC) markers. Cells were collected 48 h after PDGF-BB stimulation. Shown on the right side are representative blots from the same polyacrylamide gel. Control: vehicle + PDGF; normalization to β-actin. (B) Dose response of proliferation inhibition. CellTiter-Glo assays were performed 72 h after PDGF stimulation. The basal level reading (i.e., 72 h after adding solvent [control to PDGF-BB]) was subtracted. Control: vehicle, 72 h PDGF stimulation. Red square = tubastatin; black triangle = RGFP966. (C) Migration measured with scratch assay. Pictures show the scratch gaps before (0 h) and after (24 h) PDGF-BB stimulation. Control: vehicle + PDGF. (D) Quantitative real-time polymerase chain reaction assay. MOVAS cells were pretreated with vehicle or inhibitors for 2 h and then stimulated with tumor necrosis factor α (TNFα) for 4 h. Control: vehicle + TNFα; normalization to glyceraldehyde-3-phosphate dehydrogenase. SMA = smooth muscle actin; HDAC6 = histone deacetylase 6; IL-6 interleukin-6; MCP-1 = monocyte chemoattractant protein-1; MMP = matrix metalloproteinase; mRNA = messenger ribonucleic acid; SMHC = smooth muscle myosin heavy chain.



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