Following FACs isolation, we resuspended MuSCs in myogenic cell culture medium containing DMEM/F10 (50:50), 15% FBS, 2.5 ng ml−1 fibroblast growth factor-2 and 1% penicillin-streptomycin. We added the indicated doses (see respective Figure legends) 50 nM of SAG1.3 (Cayman Chemical Company, catalog # 11914), 1 µM of GSA-10 (Sigma-Aldrich, catalog # SML1171), 25 nM of Fluticasone (Sigma-Aldrich, catalog # F9428), 5 µM of Cyclopamine (STEMCELL Technologies, catalog #72072), 1 µM Purmorphamine (Selleck Chemical, catalog # S3042), 100 nM of Vismodegib (Selleck Chemicals, catalog # S1082) or 0.1 µg/ml SHH (R&D Systems, catalog # 461-SH-025) to MuSCs cultured on collagen coated dishes for the first 24 h. The cells were then trypsinized and cells reseeded onto hydrogels for an additional 6 days of culture. All treatments were compared to their solvent (DMSO) vehicle control.
For IFT88−/− MuSCs transplant studies, we infected MuSCs with lentivirus encoding elongation factor-1α promoter-driven luc-IRES-GFP (GFP/luc virus) for 24 h in culture as described previously6 (link). Cells were assayed for GFP 48 h post-infection using an inverted fluorescence microscope (Carl Zeiss Microimaging).
For IFT88−/− MuSCs transplant studies, we infected MuSCs with lentivirus encoding elongation factor-1α promoter-driven luc-IRES-GFP (GFP/luc virus) for 24 h in culture as described previously6 (link). Cells were assayed for GFP 48 h post-infection using an inverted fluorescence microscope (Carl Zeiss Microimaging).
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