Figure 1. NICD1 Rescues the Loss of Pax7^CE/f Satellite Cells.

(A) Schematic describing the alleles used and the genetic approach used to activate NICD in Pax7^CE satellite cells. CreERT2 driven from the Pax7 promoter simultaneously promotes the excision of exon 2 of Pax7 and the expression of the Notch intracellular domain (NICD1) from the Rosa 26 locus. NICD cells can be traced by the expression of iRES-GFP. See also Figure S1.

(B) Schematic of the tamoxifen regimen used in this study. 6-7 week old mice were given tamoxifen via IP injection for 5 consecutive days. Satellite cells were analyzed 2 weeks after the last tamoxifen injection.

(C) Graph shows the number of Pax7⁺ satellite cells per EDL fiber analyzed immediately after isolation. Data are shown as mean ± SEM. n=3; >50 fibers per genotype were counted. ***p≤0.001.

(D) Quantification of the number of satellite cells per EDL fiber. n=6; >50 fibers per genotype were counted. ***p≤0.001.

(E) Immunofluorescence staining of satellite cells on EDL myofibers stained for α7-Integrin (red) and GFP (green). Nuclei were counterstained with DAPI (blue). GFP is detected only in NICD⁺ satellite cells. Insets show cropped image of single satellite cells. Scale bar represents 100μm.

(F) Representative immunofluorescence staining showing lack of GFP expression in Pax^CE/+:Rosa^+/+ satellite cells (upper panel). Pax7 and GFP were detected in Pax7^CE/+:Rosa^Notch satellite cells (middle panel). Pax7^CE/f:Rosa^Notch satellite cells expressed GFP but not Pax7 (lower panel). Pax7 is shown in red; GFP is shown in green; and nuclei were counterstained with DAPI (blue). Scale bar represents 20μm.

(G) Graph shows the number of Pax7⁺ and GFP⁺ cells per EDL fiber isolated from mice of the indicated genotype. n=3; > 50 fibers per genotype were counted.