Fig 3. Effect of GPR43 on the expressions of myogenic genes and proteins and nuclear localization of MEF2A, PGC-1α, and NFATc1 in L6 myotube cells.

L6 myotube cells were treated with 50 nM GPR43-specific siRNA for 24 h as described in the Materials and Methods. Total RNA was extracted from the cells after the treatment or no treatment with 0.5 mM acetic acid for 30 min and with or without 2 mM araA preincubated for 20 min. Next, quantitative real-time PCR analysis was carried out for determinations of GPR43 gene expression (A), expressions of mef2a, mb, slc2a4, ppargc1a, cycs, and sdha genes (B). Muscle proteins, pAMPK, myoglobin, GLUT4, MEF2A, PGC-1α, pNFATc1, NFATc1, and CaMKKβ were extracted and analyzed by western blotting as described in Materials and Methods (C). Nuclear fraction and cytosolic fraction were separated from GPR43-specific siRNA transfected or non-transfected L6 cells after the treatment or no treatment with 0.5 mM acetic acid for 30 min. Nuclear and cytosolic proteins were examined by western blotting as described in Materials and Methods (D). Each bar represents the mean ± SE (n = 3–6). Multiple comparisons were analyzed with one-way ANOVA followed by the Tukey-Kramer post hoc test. Statistical differences are shown as*p< 0.05, **p<0.01, compared with non-treated control; ^#p< 0.05, ^##p<0.01, compared with GPR43 siRNA; ^§p< 0.05, ^§§p<0.01, compared with GPR43 siRNA + ace; ^op< 0.05, ^oop<0.01, compared with araA + ace.