Phase-contrast microscopy, immunofluorescence staining, and qPCR analysis were performed as in [12 (link)]. Cell counting for Hoechst (Sigma–Aldrich, St. Louis, USA, B-2883) and T (Abcam, Toronto, Canada, AB20680) nuclear staining was performed as in [129 (link)]. Primary antibodies against PAX7 (Developmental Studies Hybridoma Bank, Iowa City, USA, AB528428), MKI67 (KI67)(Abcam, AB15580), MYOD1 (Abcam, AB133627), and secondary Cy3 goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, USA, 111-165-003), AlexaFluor546 goat anti-mouse IgG1 (ThermoScientific, Waltham, USA, A21123), and AlexaFluor647 goat anti-rabbit IgG (ThermoScientific, A21244) antibodies were also used. The antibody staining for PAX7, MYOD1, and KI67 were conducted similar to [13 (link)], with the exceptions that permeabilization was carried out with 0.5% TritionX-100 and 100 mM glycine in Tris-buffered saline (TBS), blocking solution was 2% bovine serum albumin (BSA), 5% goat serum, and 0.1% Tween20 in TBS, and the stains were mounted with PermaFluor (ThermoScientific, TA-006-FM). CellProfiler 3.0 was used to analyze the PAX7, MYOD1, and KI67 staining.
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