Figure 6. Notch activation in myofiber upregulates Notch ligands’ expression, therefore stimulates Notch-activation and self-renewal of satellite cells niched on MCK-N1ICD myofibers.

(A–C) Immunofluorescence images (A) and quantification of Pax7⁺ cell numbers in CTX-damaged (B, n = 3) and dystrophic TA muscles (C, n = 7). (D) Western blot results of Pax7 in muscle protein extracts. (E) Immunofluorescence image of one EDL myofiber to show that Pax7 cells are not from MCK-Cre lineage (Td ^–) of MCK-N1ICD mice. Arrow points to Pax7⁺/Td^– cell, arrow-head points to Pax7^–/Td⁺ myonucleus. (F) Relative mRNA levels of Notch ligand genes. (G,H) Immunofluorescence images (G) and quantification result (H, n = 3) of intact EDL myofibers. Arrow points to Dll4 immuno-signal patch on myofiber. (I,J) Immunofluorescence images (I) and quantification (J, n = 4) of GFP percentage in satellite cells (Pax7) on EDL myofibers. Arrow in I points GFP^–/Pax7⁺ satellite cells; black and white images to shown individual channels of the outlined area on left images. *p<0.05, **p<0.01. Bar graphs indicate mean SEM.

DOI: http://dx.doi.org/10.7554/eLife.17355.015

Figure 6—figure supplement 1. Real-time PCR results of Notch-related gene expression in primary myoblasts (A, n = 3) and myofibers (B, n = 5).

*p<0.05, **p<0.01, ***p<0.001.

Values are mean SEM.

DOI: http://dx.doi.org/10.7554/eLife.17355.016

Figure 6—figure supplement 2. Expression of Notch pathway genes in myofiber.

(A) Experiment design. (B) Immunofluorescence images of EDL myofiber from Pax7^nGFP mice before and after trypsin stripping. Nuclear GFP labels satellite cells and DAPI labels nuclei. (C) Gel electrophoresis images of RT-PCR results using mRNA isolated from EDL myofibers. (D) Immunofluorescence staining of Pax7 and Dll4 in EDL myofibers. (E) Western blot results of WT myofibers after trypsin-stripping, showing an expression of activated Notch1 (N1ICD) and its target Hes1.

DOI: http://dx.doi.org/10.7554/eLife.17355.017