Histone h3k9ac

5 × 10⁴ cells were plated in 6-well plates and grown with the conditions and drug concentrations as described for the proliferation assay. Cells were harvested at 48 h and resuspendend in protein loading buffer. After sonication the samples were centrifuged at 14,000 rpm in an Eppendorf microcentrifuge (Eppendorf, DE) and the supernatants were analyzed by western-blot. The following antibodies were used: histone H3 (Cell Signaling #9715), histone H3K9ac (Cell Signaling # C5B11), GAPDH (Cell Signaling #D16H11), p53 (ThermoFisher #PA527822), and p53K382ac (Abcam 75754). Quantification was carried out using imageJ.

Eight confluent 10-cm dishes of PDGFR-β⁺ cells or SMCs (~8 × 10⁶ cells) were treated with 1% formaldehyde for 10 min to cross-link protein to DNA and were neutralized with glycine. Cells were scraped in cold PBS and subjected to ChIP assay using the SimpleChIP Plus Enzymatic Chromatin IP Kit (Cell Signaling) with an antibody targeting KLF4 (Abcam) or Histone H3K9Ac (Cell Signaling). Coprecipitated DNA was analyzed by qPCR with primer sets (sense and antisense) targeting the promoter locus of Foxm1 (5′-GCCGATTGGCGACGCT-3′ and 5′-CGCCGCTTTCAGTTGTTCCG-3′) or Tgfb1 (5′-CCTTGACACTCTCATCCGCA-3′ and 5′-TGGGACTTCGTGAGAAAACAGA-3′). For ChIP assay with the anti-KLF4 antibody, the amount of coprecipitated DNA was normalized to that from ChIP using control rabbit IgG (1:500, Cell Signaling). For ChIP assay with the H3K9Ac antibody, the amount of coprecipitated DNA in PDGFR-β⁺ cells is relative to the amount of PCR product amplified from input samples reserved before immunoprecipitation and then normalized to this value in SMCs.

Sphingosine, ceramide, and Sphingosine-1-phosphate were from Avanti Polar lipids, SphK1 inhibitor PF-543 from Pfizer, SphK2 inhibitor ABC294640 was from Active Biochemicals. MitoTracker Deep Red was from Life Technologies Ltd. Antibodies recognising the following proteins were used: flotillin 1 (610820), flotillin 2 (610384) from BD Transduction Laboratories, SphK1 (12071) and Isg15 (2743) from Cell Signaling, Histone-H3 K9ac (ab10812), Histone H3 K14ac (ab52946), Histone H4 K5ac (ab51997), Histone H3 (ab10799), Histone H4 (ab10158) all from Abcam, SphK2 (17096) from Proteintech Group. All experiments using antibodies for Western blotting were performed at least twice and representative blots are shown. Quantification was carried out by scanning and densitometry using Image J.