Disruption of the HSP90-STAT3 interaction by 17DMAG protected C2C12 myotubes from C26 CM-induced atrophy.
17DMAG, an inhibitor of HSP90, was then introduced in the C2C12 myotube atrophy model, which was established by the treatment of C2C12 myotube with C26 derived CM; the results showed that 17DMAG treatment prevented myotube shrinking, increased the myotube diameter, and enlarged the myotube area (Figure 3a and b, Figure S2a). Notably, 17DMAG treatment significantly restored MHC expression, decreased the STAT3 activation, and down-regulated the expression of myostatin, MuRF-1, and Atrogin-1 both at the protein and mRNA levels (Figure 3b-d). In addition, the co-expression of MHC, myostatin, and phosphorylated STAT3 (pY-STAT3) was also confirmed by immunofluorescence (Figure 3c). Importantly, 17DMAG disrupted the increase in the binding of HSP90 and JAK2 to STAT3, suggesting that the molecular chaperone activity of HSP90 might prolong the JAK2-STAT3 interaction and STAT3 activation in C26 CM-induced C2C12 cells (Figure 3e). Thus, we concluded that the 17DMAG treatment protected C2C12 myotubes from C26 CM-induced muscle atrophy and catabolism. In contrast, 17DMAG treatment was not able to ameliorate LPS induced myotube atrophy, suggesting its effect was not TLR4 dependent (Fig S2b).