Inhibition of G9a Histone Methyltransferase Converts Bone Marrow Mesenchymal Stem Cells to Cardiac Competent Progenitors

Figure 1

Effect of G9a HMTase inhibition on bone marrow cells. (a–d) Fluorescent staining of nontreated and BIX01294-treated bone marrow stem cells with DAPI for labeling all nuclei and antibody specific for dimethylated form of histone H3 at lysine 9 (H3K9). (e) Immunoblot of protein isolated from nontreated and BIX01294-treated bone marrow stem cells. Data from panels (a)–(e) demonstrate that methylation of H3K9 is reduced upon exposure to BIX01294. Blotting for GAPDH and total histone H3 verified equal amounts of protein were added for each sample. (f) Immunoblot showing that knockdown of G9a HMTase expression using either of two gene-specific shRNAs (G9a676 and G9a3291) caused decreased H3K9 dimethylation, in comparison to cultures transfected with scrambled shRNA or the empty vector. Blotting for GAPDH and total histone H3 served as controls. For all blots, side bars indicate the molecular weight of the specific proteins detected, including the 172 and 158 kD bands that correspond to the two known splice variants of G9a HMTase: G9a-L, G9a-S [20]. (g) Real-time qPCR analysis showing that G9a HMTase specific knockdown in bone marrow stem cells upregulated Mesp1 and brachyury mRNA expression, as compared to scrambled shRNA controls, which is consistent with results obtained with BIX01294 treatments. ; .