FIG. 8.

Inhibition of myogenic conversion by HDAC3. (A to C) An MyoD expression plasmid (0.4 μg) was transfected into murine C3H10T1/2 fibroblasts along with constructs expressing Flag-MEF2D (0.6 μg), HA-HDAC3 (0.3 μg), SMRTe (0.3 μg), mHDAC3i (0.3 μg), and CaMKIV-ca (0.3 μg). On day 7, myosin heavy chain (MHC) expression was detected by indirect immunofluorescence microscopy with anti-MHC monoclonal antibody and a Cy3-labeled secondary antibody. Average values of three independent experiments are illustrated in panels A and C, with some representative images shown in panel B. (D) C2C12 cells were transfected with the Flag-HDAC3 expression vector. Sixteen hours later, cells were washed once with PBS and fed with DMEM containing 2% horse serum to induce differentiation. Cells were harvested in ChIP lysis buffer on day 0 for chromatin immunoprecipitation with anti-Flag antibody to determine association of Flag-HDAC3 with the MCK promoter. An anti-HA antibody (aHA) was used as a negative control (lane 2). (E) C2C12 cells, maintained in DMEM with 20% FBS, were washed once with PBS and fed with DMEM containing 2% horse serum to induce differentiation. Cells were harvested in buffer K on days 0 and 3 to prepare extracts for immunoprecipitation with anti-MEF2D antibody and immunoblotting with anti-HDAC3 and anti-MEF2D antibodies. An anti-GFP antibody was used as a negative control for immunoprecipitation (IP) (lane 3). Western blotting (WB) with anti-HDAC3 antibody was carried out as described in the legend to Fig. 1E.