Figure 8. (−)-Epicatechin ((−)-EPI) stimulates C2C12 myotube growth via GPER activation.

Cell length and width was visualized by fluorescence microscopy (EVOS^® FLoid^® Cell Imaging Station) in formaldehyde fixed myotubes (cells with ≥2 nuclei). For visualization of cytoskeleton (F-actin filaments) and nuclei cells were stained with fluorescently-labeled phalloidin and Hoechst 33258 for 20 min at room temperature, respectively. (A) Representative fluorescence images corresponding to cells treated with either DMSO vehicle (control), 10 µM (−)-EPI or 0.1 µM G-1 (GPER agonist) with or without 0.1 µM G-36 (GPER antagonist) and 0.1 µM siRNA targeting GPER (see methods section for knockdown conditions). Scale bar = 100 µm. (B) Representative set (same treatments as in A) of fluorescence images zoomed-in. Nuclei staining were omitted for better visualization. Scale bar = 100 µm. (C) Quantification of myotubes length (µm). Data is expressed as median and interquartile range derived from at least 3 independent experiments performed each in quadruplicate. ***P≤ 0.001 vs. control as analyzed by Kruskal-Wallis test followed Dunn’s multiple comparisons test. (D) Quantification of myotubes width (µm). Data is expressed as median and interquartile range derived from at least 3 independent experiments performed each in quadruplicate. *P≤ 0.05 vs. control as analyzed by Kruskal-Wallis test followed Dunn’s multiple comparisons test. White arrows denote representative myotubes width at the wider portion of the cell.