Fig. 1: Retinol metabolism is enriched in quiescent satellite cells.
A Experimental study design. Eight to 12-week-old Pax7creER/+; Rosa26 tdTomato/+ mice were administrated with tamoxifen for five consecutive days followed by 5-day waiting period to label MuSCs with tdTomato. Quiescent and freshly isolated MuSCs were collected immediately after 5-day waiting period. MuSCs from CTX injury-, freeze injury- and overload-mouse models were harvested 3-, 4- and 7-day after surgery, respectively. Quiescent MuSCs as well as MuSCs from CTX injury-, freeze injury- and overload-mouse model representing cells in vivo state were fixed by PFA perfusion before isolation process. Freshly isolated MuSCs were collected without fixation. RNA-seq library of MuSCs sorted by FACS were constructed by Smart-RNAseq. CTX, cardiotoxin; PFA, paraformaldehyde; FACS, fluorescence activated cell sorting. B, C Representative pictures showing EdU and PAX7 staining in the sham model and different injury models. Arrows represent EdU+/PAX7+ cells; arrowheads represent EdU-/PAX7+ cells. C Quantification of EdU-positive MuSCs. n = 3 mice/treatment, >450 Pax7+ cells counted/mouse. D Principal component analysis (PCA) of the gene expression in quiescent MuSCs, CTX-injury MuSCs, freeze-injury MuSCs, overloaded MuSCs and FISCs. FISCs, freshly isolated satellite cells. n = 2 biological replicate RNA-seq data/group. E–G Expression heatmap of (E) quiescence-, (F) Notch signaling pathway- and (G) stress-associated genes induced by the isolation process. n = 2 biological replicate RNA-seq data/group. H Venn diagram of differentially downregulated genes in different activated MuSCs compared to quiescent cells. The red dashed area represents quiescence-maintaining genes. I Kyoto Encyclopedia of Genes and Genomes pathway analysis of quiescence-maintaining genes in H. The top 12 categories are shown. J Heatmap of representative gene expression for the retinol metabolism signaling pathway. n = 2 biological replicate RNA-seq data/group. Scale bar: 50 µm in B. Statistical results are expressed as the mean ± SEM. ***p < 0.001.
