Fig. 5. Transient expression of Lbx1 in transfected C2C12 cells prevents myotube formation. (A) Immunochemical detection of Lbx1 protein in C2C12 cells. In control cells (pCX-EGFP), no Lbx1 can be detected. In cells transfected with the pCX-Lbx1 construct, Lbx1 is expressed in cells cultured in growth medium and 5 days after serum withdrawal (arrowheads). Bars, 25 µm. (B) Phase-contrast microscopy of Lbx1-expressing and control C2C12 cells. C2C12 cells were transfected with either pCXLbx1 or pCXEGFP (control). Both cells are morphologically indistinguishable when cultured in C2C12 growth medium (Upper panels). When induced to differentiate by serum withdrawal, Lbx1-expressing C2C12 cells exhibit abnormal myotube formation; numerous cells fail to fuse and remain as mononucleated cells. Even if myotubes are formed, they are extremely short, whereas control cells differentiated into myotubes after 5 days under serum-free conditions (Lower panels). Bars, 100 µm. (C) Immunochemical analysis. To observe details of forming myotubes, PCX-EGFP-transfected (control) and pCX-Lbx1-transfected cells were immunostained for myosin heavy chain (MHC, red) and counterstained with Hoechst dye 33342 (blue). Bars, 100 µm. (D) Ultrastructural analysis of myotubes formed in vitro. The sarcomeric organization with Z bands (arrow) is evident in pCX-EGFP-transfected cells. In C2C12 cells transiently expressing Lbx1 (pCX-Lbx1), muscle fibers do not contain a rudimentary sarcomeric organization with Z bands. Bars, 1 µm. (E) Number of nuclei of in-vitro-formed myotubes. At 120 hours after serum withdrawal, the average number of nuclei in MHC-positive myotubes was counted. The myotubes formed from control cells (pCX-EGFP) contain 6.9±1.5 nuclei (n=2002), whereas those from Lbx1-expressing C2C12 cells (pCX-Lbx1) contain only 1.7±0.2 nuclei (n=644). This difference was statistically significant as calculated using Student's t-test (P<0.05). (F) Results of cell counting. No significant differences were found in the numbers of cells of Lbx1-expressing C2C12 cells and control cells comparing 35-mm culture dishes. , Lbx1-expressing cells; , control cells. (G) Rate of cells with nuclear condensation. Lbx1-expressing C2C12 and mock-transfected control cells were stained with Hoechst dye 33342, and the number of cells with nuclear condensation was counted. The numbers of cells examined were as follows. White bars (Control): D0, n=3908; D3, n=9847; D5, n=10,763. Gray bars (Lbx1-transfected): D0, n=3501; D3, n=6180; D5, n=6005.