Figure 3. Administration of HSP90 inhibitor 17DMAG prevented C26
CM-induced myotube atrophy and disrupted the binding between HSP90 or
JAK2 with STAT3.
(a) C2C12 myotubes were treated with C26 CM, C26 CM plus HSP90
inhibitor 17DMAG (0.1 μmol), or normal media for 24 h. H&E staining
shows the morphological changes in C2C12 myotubes. Scale bar: 50 μm.
(b) The histogram represents the average myotube diameter (μm)
and myotube area (μm2) in control, CM-treated, and
17DMAG plus CM-treated C2C12 myotubes. The number of myotubes was
quantified using ImageJ. qRT-PCR analysis of atrogin-1 and MuRF-1 in the
gastrocnemius muscles of mice (b, right part). (c) C2C12 cells
were immunostained with the MHC (MF20) antibody. MHC, green, DAPI, blue.
The expression and subcellular localization of myostatin and pY-STAT3 in
C2C12 myotubes were detected by immunofluorescence staining. Myostatin
and pY-STAT3, green, PI, blue. Scale bar: 30 μm. (d) The
expression of MHC, HSP90, pY-STAT3, atrogin-1, and MuRF-1 in control,
CM-treated, and 17DMAG plus CM-treated C2C12 myotubes was analyzed by
western blot, GAPDH served as a loading control. (e) The
lysates of C2C12 cells treated with C26 CM or DMEM (control) were
immunoprecipitated with the STAT3 antibody, the HSP90 antibody, or
control rabbit IgG. The immunopellets were detected by immunoblot
analysis with the anti-HSP90 antibody and the anti-STAT3 antibody. Data
represent the mean ± standard deviation (SD). *p<0.05,
**p<0.01, ***p<0.001, ****p<0.0001 denotes
compared to control; #p<0.05, ##p<0.01,
###p<0.001 denotes compared to C26 CM treated myotubes.