Fig. 3

Loss of PYGM function delays C2C12 cell differentiation. A Two sgRNAs designed to target the first exon of PYGM. The resulting allele harbors a 235-bp deletion in exon 1. B Schematic illustration of the surveyor primers used to identify PYGM-knockout clones. C PYGM-knockout clones (1, 2, 3, 4) were validated by RT-PCR. Wild-type clones (1, 2, 3, 4) served as controls. D PYGM-knockout clone verified by DNA sequencing. E Western blotting analysis to verify PYGM-knockout clones (KO-1, KO-2). Wild type clones (WT-1, WT-2) served as positive controls. β-actin served as an equal-loading control. F Representative images of immunostaining for MyoG (green) or MHC (green) in PYGM-knockout (KO-1, KO-2) and wild-type (WT-1, WT-2) C2C12 cells cultured in differentiation medium for 24 h (MyoG) or 48 h (MHC). DAPI (pseudo-colored red) served to visualize nuclei. Scale bars, 100 μm. G Numbers of MyoG-positive (MyoG⁺) cells per view described in (F). H Relative expression of MyoG in the cells described in (F), as determined by RT–qPCR. I Fusion index calculated in the cells described in (F). J Relative expression of MHC in the cells described in (F), as determined by RT–qPCR. All images in the figure are representatives of three independent experiments. Values presented indicate the means ± SE obtained from three independent experiments