Tmp269

HASMCs were obtained from Cell Applications (#355–75a). To induce calcification in HASMCs, cells were treated with DMEM supplemented with 10% fetal bovine serum, 10 mM β-glycerophosphate disodium, 50 μg/mL L-ascorbic acid, and 10 nM dexamethasone, as previously described⁴⁸ . To assess the effects of HDAC9 inhibition on calcification, cells were treated with either 100 nM TMP269 (Selleckchem, Cat No. S7324) or vehicle (DMSO) control. Media and treatment were replenished every 24 hours for 7–10 days. Either RNA was isolated from cells to assess mRNA levels (see below) or the cells were fixed in 10% formalin and incubated with Alizarin Red or von Kossa stain to detect calcification. For Alizarin Red staining, cells were treated with a 2% Alizarin Red solution (pH 4.1–4.3) for 20 minutes, followed by multiple washes with distilled water. For von Kossa staining, cells were incubated in 5% silver nitrate solution and exposed to a 100 Watt bulb for 1–2 hours. Removal of unreacted silver was performed by subsequent treatment with 5% sodium thiosulfate. Cells were then washed with distilled water.

For the HDAC inhibition assay, cells were pretreated with TSA (5nM, Sigma-Aldrich). For the TNF-α secretion assay, human THP1 or murine macrophage RAW264.7 cells were treated with TSA, VPA (0.1 μM), Ms275 (5 μM), NIC (0.5 μM) HPOB (0.5 μM) (Sigma-Aldrich), TMP269 (0.1 μM), PCI-34051 (0.1 μM), RGFP966 (0.5 μM) (Selleckchem), paragyline (3 μM), 5'azacytidine (10 μM) or anacardic acid (10 μM) (Cayman Chemical). The concentration of HDAC inhibitors used did not induce cytotoxicity (Supplementary Fig.5 a-h). It is important to note that at high concentrations, HDAC inhibitors blocked TNF-α secretion (Supplementary Fig. 7b-i) ^{30 (link)}.