Figure 5. Inhibition of histone modifiers HDAC1 and EZH2 rescues OB differentiation of MM-exposed MC4 cultures.

(A–F) MC4 cells were exposed to 5TGM1 MM cells as diagrammed in Fig 1A in the absence of inhibitors. After MM removal at d0, the MC4 were cultured in OB differentiation media for 2 to 4 days with either DMSO vehicle, MC1294 (10 μM), or GSK126 (5 μM) added as indicated. (A,B) The effects of the inhibitors (A) MC1294 (HDACi) and (B) GSK126 (EZH2i) on global levels of H3K9ac, H3K27me3, H3, HDAC1, EZH2 levels in MC4 cells on day 2 were assessed by Western blot using antibodies as indicated. (C–F) Effects of the inhibitors MC1294 and GSK126 on (C) Runx2, (D) Ocn, (E) Bsp, and (F) Alpl mRNA expression during differentiation of control and 5TGM1 MM-exposed MC4 at day 0 (no inhibitor) or after 4 days of differentiation (d0±MM, d4±MM). Error bars represent SEM for 3 biological replicates. (G) Human MM1.S MM cells in transwells (or empty control transwells) were co-cultured with MC14 cells for 72 h. Following transwell removal, the MC14 cells were cultured in osteogenic media +/- GSK126 (2.5 μM) and mineralization was assessed using Alizarin Red staining at d21; the GSK126 was absent d14–21. Shown is density quantitation for the average of 6 wells with SEM and significance indicated.