miR669a overexpression inhibits myogenic differentiation in Sgcb-null cardiac progenitors. (A) qPCR analysis for MyoD expression in wt transduced Sgcb-null (KO + LVbSG) and Sgcb-null clones (KO; top). Western blot analysis for SGCB expression in differentiating wt, transduced Sgcb-null (KO + LVbSG), and Sgcb-null clones (KO; bottom). (B) Sgcb expression did not interfere with the ability of transduced Sgcb-null cardiac progenitors (+LVbSG) to form myotubes in serum starvation condition similar to not transduced cells (KO). (C) Time course of ⁴⁵Ca²⁺ uptake into wt, transduced Sgcb-null (red line), and Sgcb-null cells (black line). (D) qPCR analysis for miR669a (top) and Sfmbt2 (bottom) expression in Sgcb-null (KO) and wt clones. Analysis was performed on three independent experiments. (E) miR669a expression in proliferating (prolif) and differentiating (white bars) wt and Sgcb-null (KO) cardiac clones.(F) Western blot analysis for YY1 expression in Sgcb-null (KO) and wt cardiac progenitors (top), in Sgcb-null progenitors treated with E64 for 48 (KO+E64/48h) and 72 (KO+E64/72h) h, and in Sgcb-null progenitors transduced with LVbSG (bottom). (G) Relative (rel.) expression of miR669a (top) and Sfmbt2 (bottom) in Sgcb-null cardiac progenitors treated for 48 and 72 h with 100 µM E64 (white bars) and untreated (black bars). (H) miR669a (top) and Sfmbt2 (bottom) expression analysis in wt and in Sgcb-null cardiac progenitors transduced with LVbSG or LV-EGFP (KO). Ct, cycle threshold. (I) TaqMan assay analysis for miR669a and miR669q expression (top) and qPCR analysis for MyoD expression (bottom) in wt cardiac progenitors after transfection with scrambled (scr) miRNA and miR669a LNA knockdown (LNA). miR669a and miR669q silencing activated MyoD expression. (J) MyoD immunofluorescence analysis in the wt cardiac clone (J8 Ven WT) treated with scrambled miRNA (top) and with miR669a LNA (bottom). High magnification of MyoD-positive cells is shown in the insets. (K) miR669a (top) and MyoD (bottom) expression analysis in the Sgcb-null cardiac clone (KO) transfected with scrambled miRNA and miR669a. miR669a dramatically reduced MyoD expression in the miR669a-transfected Sgcb-null clone. (L) Immunofluorescence analysis for MyoD expression in the Sgcb-null cardiac clone transfected with scrambled miRNA (top) and miR669a (bottom). Three independent experiments were performed in triplicates and statistically analyzed using Student’s t test; P < 0.05 in all panels. Error bars show means ± SEM. Bars, 50 µm.