FIG. 10.

Glypican-1 is required on the plasma membrane for a proper muscular differentiation process independent from extracellular matrix-associated glypican-1. C2C12 myoblasts were transiently cotransfected with scrambled shRNA (shCtrl) and a plasmid containing the sequence for E-GFP (A, D, and I) or shGly (B, E, C, and F). At 48 h after transfection, the myoblasts were induced to differentiate for 2 days (A, B, D, and E) or 4 days (C, F, and I). The cells were fixed and analyzed by immunofluorescence for glypican-1 (red) (A, B, and C), myogenin (red) (D and E), or myosin (red) (F and I). Panels G and H present the same images as panels D and E, respectively, but without the E-GFP signal, in order to better visualize myogenin nuclear staining. The arrowheads indicate the ECM-associated glypican-1. The arrows indicate the nuclei of transfected cells. (J) Shown on the left is a quantification of the cotransfected myoblasts (shCtrl/E-GFP or shGly/E-GFP) containing myogenin-positive nuclei compared to the total cotransfected cells (E-GFP positive) after 2 days of differentiation of 10 random fields. Shown on the right is a quantification of the E-GFP-myosin-positive myotubes compared to the total number of E-GFP-expressing cells after 4 days of differentiation. The data correspond to the means ± standard errors of the results obtained with 10 random fields.