Fig. 5

Overexpression of Stx4 can override the block of myoblast differentiation in Cdo-depleted cells. a, b C2C12 cells were transfected with control, Stx4, or shStx4 expression vectors, and the lysates were analyzed for the levels of phospho-p38MAPK (p-p38) relative to total p38MAPK (p38). The relative levels of p-p38 are quantified and added under each well. c C2C12/pSuper and C2C12/Cdo shRNA cells were transiently transfected with pcDNA or Stx4 expression vector, plus GFP expression vector to mark transfectants. Confluent cultures were then fixed and stained with antibody to p-p38 (red). Cell nuclei were visualized by staining with DAPI (blue). The white arrows in p-p38 panels mark the transfected cells. Size bar = 10 μm. d Quantification of cultures shown in c. GFP⁺ cells were scored as positive or negative for p-p38 staining. These experiments were repeated three times with similar results. Significant difference from control, *p < 0.01. e Quantification of the relative signal strength of p-p38 in GFP-positive cells. Values are determinants of 10 fields and experiments were repeated three times with similar results. *p < 0.01. f C2C12/pSuper and C2C12/Cdo shRNA cells were transiently transfected with pcDNA or Stx4 expression vector, and the lysates were analyzed for the levels of phospho-p38MAPK (p-p38) relative to total p38MAPK (p38). The relative levels of p-p38 are quantified and added under each well. g C2C12 cells were transfected with pSuper or shStx4 expression vectors and were immunoprecipitated with control IgG or anti-Cdo antibody and immunoblotted with antibodies to JLP, Bnip2, Cdo, Stx4, and pan-Cadherin as a loading control. h Similar sets of cells as shown in panel c were induced to differentiate for 3 days and immunostained for MHC and GFP expressions. Size bar = 100 μm. i Quantification of myotube formation from panel e. GFP⁺ myotubes were quantified for MHC expression and the number of nuclei present in myotubes. Values are determinants of more than 10 fields and these experiments were repeated three times with similar results. Significant difference from control, *p < 0.01, **p < 0.005