Metformin reduces autophagy in developing myotubes. A, B) Confocal microscopy revealed that metformin treatment reduced autophagosome (green LC3-II labeling; beveled arrowheads) and lysosome (red lysotracker labeling; arrowheads) density, with no significant difference in colocalization (arrows) (n=45). Scale bar = 20 μm. C, D) TEM also revealed a reduction in autophagosome density (n=30). E) Relative expression of LC3b-II was also reduced following metformin treatment. There was insufficient total protein staining near the 15 kDa molecular weight marker, so LC3b-II was normalized to total protein from the 50–100 kDa range. n=6; *P≤0.05, ** P≤0.01.