Figure 3.

Cultured dKO Cells Show Evidence of Altered Myogenic Programming

(A–X) Cultured dKO satellite cells adopt a fibroblast-like appearance but continue to express PAX7 and desmin (DES). (A–D) Phase images of dKO and dHet satellite cells after 3 or 7 days in culture. (E–L) Immunofluorescence analysis of PAX7 expression in dHet (E, F, I, and J) and dKO (G, H, K, and L) satellite cells after culturing for 3 (E–H) or 21 (I–L) days in GM. Occasional PAX7– cells were observed at day 21 (arrows) (K and L). (M–P) dKO cells maintained PAX7 expression after 21 days in DM. In dHet cells (M and N), PAX7 was downregulated in myotubes (arrows), but maintained in mononuclear cells (examples at arrowheads). Arrows in (O) and (P), example of an occasional PAX7– dKO cell. (Q–X) Comparison of DES expression in dHet (Q, R, U, and V) and dKO (S, T, W, and X) satellite cells 3 or 14 days in GM. dKO cells lacking detectable DES were common by day 14 (X), arrows.

(Y–B′) dKO satellite cells show a propensity for adipogenic differentiation. dKO cells were cultured in GM for 7 days and switched to DM for 13 (Y and Z) or 21 (A’ and B’) days. Approximately 10% of cells accumulated lipid droplets and were PLIN1+; examples at arrows in (Y and Z). Inset in (Z), phase contrast image of boxed lipid-filled cell in (Y) and (Z). PAX7 is downregulated in adipogenic cells; arrows in (A’) and (B’). Exposure times (ms) are shown in some panels to allow comparisons of apparent expression levels between genotypes. Scale bars represent 50 μm.