Mesenchymal-lineage C2C12 cells were maintained and routinely passaged as previously described (28 (link), 29 (link)). A mouse Opg promoter-luciferase construct, containing 1.6 kb of promoter sequence spanning from −1486 to +133 bp relative to the Opg transcription start site, was obtained as a kind gift from Dr. Malayannan Subramaniam and Dr. John Hawse at the Mayo Clinic (30 (link)). For analyses of Opg transcriptional activity, cells were transfected with Lipofectamine (Invitrogen) and a DNA mixture consisting of 300 ng of this mouse Opg promoter construct, promoterless Renilla luciferase (pRL-null) (10 ng), and either pCMV-Hdac3 expression or control plasmids (100 to 500 ng) in 12-well plates. Following an overnight incubation at 37°C, cells were treated with 10 µM SAHA or vehicle (DMSO) for 24 hours. Luciferase activity in 20 µL of cell lysate was measured using the dual luciferase assay system (Promega) and a Glomax 96 microplate luminometer (Promega) or a BioTek Synergy HT plate reader (BioTek, Winooski, VT, USA). All transfections were performed in triplicate, and data were normalized to the activity of Renilla luciferase. We also re-analyzed our previously reported ChIP-sequencing dataset (28 (link)) from mesenchymal lineage MC3T3 cells to examine the effects of the Hdac inhibitor vorinostat (10 µM) on acetylation of histone H4.