Expression of late muscle differentiation markers is affected in differentiated M19-deficient C2C12 cells.

<p>(<b>A</b>, <b>B</b>) C2C12 myoblasts were transfected with a control siRNA (si control) or a M19-specific siRNA (si M19) and were then placed in differentiation medium for 7 days. Protein extracts from transfected cells grown in proliferation medium (d0) or in differentiation-promoting conditions for 3, 5 and 7 days (d3, d5, d7) were analyzed by Western immunobloting using the M19-specific P70612 antibody (<b>A</b>) and a MHCII antibody (<b>B</b>). Densitometry analysis of the detected bands is presented as the relative expression of M19 (<b>A</b>) and MHCII (<b>B</b>) normalized to tubulin. Results are the mean ± SEM of three independent experiments. (*) and (**) indicate statistical significance at p<0.05 and at p<0.01. In a similar experiment, C2C12 myoblasts were transfected with the M19-specific shRNA vector allowing the expression of the specific shRNA with GFP. Cells were placed in differentiation medium for 7 days. Fluorescence microscopy allows the direct visualization of GFP-labeled cells expressing the M19-specific shRNA (<b>C, merge; green</b>) and the detection of MHCII using an anti-MHCII antibody (<b>D, merge; red</b>). (<b>E</b>) Protein extracts from control shRNA-transfected C2C12 cells (sh control) and M19-specific shRNA-transfected C2C12 cells (sh M19) grown in differentiation-promoting conditions for 7 days were analyzed by Western immunoblotting. The expression of the late muscle differentiation markers α-actinin 2, troponin T, MHCI and MHCII is shown, as well as the control protein tubulin.