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. 2020 May 19;18(5):267. doi: 10.3390/md18050267

Figure 4.

Figure 4

Effect of CYC31 on GLUT4 translocation: (A–C) The protein level of GLUT4 in the plasma membrane (A), cytosol fractions (B), and whole cell lysates (C) C2C12 myotubes were serum-starved overnight and treated with indicated concentrations of CYC31 for 8 h or 10 nM insulin for 5 min in serum-deprived DMEM; then, cells were lysed and membrane proteins and cytosol fractions were extracted using the Membrane and Cytosol Protein Extraction Kit (Beyotime, Shanghai, China). The protein level of GLUT4 was detected by immunoblotting. (D) Relative expression level of GLUT4 on cell membrane after CYC31 treatment: Band density was measured by Image J software and normalized to β-actin. Data was shown as mean ± SD values (n = 3), ** p < 0.01, *** p < 0.001 compared with the DMSO-treated group (E) CYC31 promotes glucose uptake in C2C12 myotubes. C2C12 myotubes were treated with the indicated concentrations of CYC31 for 8 h or 10 nM insulin for 5 min in serum-free DMEM; then, 200 μM 2-NBDG was added for 20 min in glucose-free DMEM. The fluorescence intensity was determined at ex/em 465/540 nm using a microplate fluorometer. The results shown are means ± SD (n = 5). * p < 0.05, *** p < 0.001 versus the DMSO-treated control group.