Figure 4
FOXO4 silencing rescued WNK1 silencing induced muscle cell atrophy and atrogene upregulation. (A) Immunoblots showing the siRNA knockdown efficiencies of WNK1 and FOXO4 in C2C12 cells. (B) Quantification of MAFbx, MuRF1, Bim, Noxa, and bcl6 in siRNA-treated C2C12 cells by real-time polymerase chain reaction analysis (n = 4 or 6 per experimental group). The upregulation of these transcripts induced by WNK1 silencing was cancelled upon co-transfection of Foxo4-targeted siRNA. mRNA levels were normalized against those of β-actin. (C) Immunofluorescence with a myosin heavy chain (MHC) antibody in C2C12 cells that were pre-treated with siRNA and incubated in differentiation medium for 4 days. Myotube atrophy induced by WNK1 silencing was substantially rescued by co-silencing of FOXO4. Scale bar, 200 μm. (D,E) Histograms showing proportions of myotubes according to the diameters. The shift to a higher proportion of atrophic myotubes upon WNK1 silencing was completely reversed by FOXO4 silencing (D), as was the reduction of the mean myotube diameter (E) (n = 5 or 6 per experimental group). (F) The decrease in the fusion index, indicating impaired myogenesis, induced by WNK1 silencing was also cancelled by FOXO4 silencing (n = 5 or 6 per experimental group). Values are presented as the mean ± standard error of the mean. *P < 0.05; **P < 0.01. Small interfering RNA, siRNA; WNK, with-no-lysine (K); FOXO4, forkhead box protein O4; MHC, myosin heavy chain; MAFbx, muscle atrophy F-box; MuRF1, Muscle RING-finger protein-1. Full-length blots are presented in Supplementary Fig. 10.
