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Fig. 7. Geldanamycin treatment of C2C12 myocytes triggers accumulation of myosin maturation intermediates. (A) Control, untreated C2C12 myocytes fixed with cold methanol 52 hour after induction of differentiation and stained with mAb F59. Under these conditions, F59 stains all the muscle myosin, folded and unfolded. (B) Accumulation of myosin trapped in maturation intermediates in myocytes that were treated for 22 hour with geldanamycin (GA) is revealed after methanol fixation and F59 staining. Myosin maturation intermediates accumulate in all myosin-expressing cells. (C,D) Paraformaldehyde fixation of cells treated for 22 hour with geldanamycin reveals the association of the partially folded myosin (F59 staining) with Hsp90 (anti-Hsp90 staining). (E) Control C2C12 myocytes 92 hour post-induction fixed with cold methanol and stained with mAb F59. The large, well differentiated myotubes are filled with striated myofibrils. (F) Geldanamycin-treated cells 40 hour after washing out the drug show reversal of drug effects and recovery of the myofibril formation. These cells were also fixed with methanol and stained with F59 to detect both the folded and unfolded myosin.
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