Figure 3. Z-disk formation in differentiated myotubes.

(A) Z-disk formation was analyzed in Plec^+/+ and Plec^–/– myotubes (differentiated for 10 days) by immunolabeling using antibodies to α-actinin. Plec^–/– myotubes displaying dissolution of sarcomeric structures (top image) or intact Z-striations (middle image) are shown. Scale bars: 10 μm. (B) Statistical evaluation of intact sarcomeric structures observed in myotubes differentiated for 5 (Plec^+/+, n = 426 myotubes; Plec^–/–, n = 279 myotubes), 10 (Plec^+/+, n = 330 myotubes; Plec^–/–, n = 384 myotubes), or 15 (Plec^+/+, n = 353 myotubes; Plec^–/–, n = 274 myotubes) days. Mean ± SEM, 4 experiments. (C) Statistical evaluation of intact sarcomere formation in primary myotubes differentiated for 10 days (Plec^+/+, n = 87 myotubes; Plec^–/–, n = 164 myotubes). Mean ± SEM, 3 experiments. (D) Immunoblotting of cell lysates prepared from Plec^+/+ or Plec^–/– myoblasts that were either undifferentiated (day 0) or differentiated for 5, 10, or 15 days. Antibodies used for detection are indicated. GAPDH was used as loading control. (E) Plec^–/– myotubes expressing P1d-EGFP were differentiated for 10 days and immunolabeled using antibodies to desmin and α-actinin. Note that Z-disk formation was restored and desmin was recruited to sarcomeres. Scale bar: 10 μm.