Fig. 7. GC and SM UNC-45 have different functions in muscle differentiation. C2C12 skeletal myocytes were treated for 8 days with none (0), negative control (C), GC UNC-45 antisense (G) or SM UNC-45 (S) antisense oligonucleotides. (A) SM UNC-45 antisense treatment specifically reduced SM UNC-45 mRNA expression by 50%. Northern blot of 10 µg total RNA from myotube-containing cultures treated for 8 days, labeled with a SM UNC-45 and a GAPDH probe. SM UNC-45 mRNA was expressed at control levels in the GC UNC-45 antisense-treated cultures, reflecting the presence of short myotubes. (B) Muscle differentiation was assessed by immunostaining with EA53 antibody to sarcomeric -actinin. Nuclei were stained blue with DAPI. Unstained cells appear black with blue nuclei. Robust, multinucleated myotubes were observed in cultures treated with the negative control oligonucleotide. Cultures not treated were indistinguishable from these. SM UNC-45 antisense-treated cultures had fewer myotubes than controls. The majority of differentiated cells in GC UNC-45 antisense-treated cultures were short myotubes with one to four nuclei, suggesting that GC UNC-45 is necessary for normal levels of myoblast fusion. Bar, 100 µm. (C) Subnormal levels of SM UNC-45 reduced the extent of striated myofibrils, as shown by this high magnification view of sarcomeric -actinin staining in myotubes from negative control and SM UNC-45 antisense-treated cultures. Bar, 10 µm. (D) Graphical representation of the effects of GC and SM UNC-45 antisense treatment on myotube differentiation in vitro. Bar colors are: stippled for no treatment; white for control oligonucleotide; black for SM antisense; and grey for GC antisense. Six hundred cells were counted for each sample; those not represented here were mononuclear. GC UNC-45 appears to function in cell fusion while SM UNC-45 has a role in fusion and formation of striated myofibrils within myotubes.