Supplementary Figure 3: Analysis of the hindlimb muscles lacking Mib1 in myofibers.

(a–c) The expression of Mib1 (a; n = 5 animals for each group; data are mean ± s.d.; two-sample t-test; ^∗P < 0.05), IHC staining for Mib1 (b; scale bar, 25 μm), and myonuclei number (c; n = 5 animals for each group; Data are mean ± s.d.; Poisson’s general linear model regression; N.S. not significant) in MFs from 8-week-old Mib1^WT and Mib1^ΔMFextensor digitorum longus (EDL) muscles. Myonuclei numbers in Mib1^WT and Mib1^ΔMF MFs were comparable. Statistical source data for a and c is provided in Supplementary Table 2. (d) Morphometric quantification of cross-sectional area validated in Laminin-stained TA muscles, which is comparable in Mib1^WT and Mib1^ΔMF mice;n = 5 animals for each group; data are mean ± s.d.; χ² test. (e) IHC staining for MyoD and Ki67 in TA muscles from Mib1^WT and Mib1^ΔMF mice at the indicated ages. Arrows indicate MyoD⁺Ki67⁺ cells; scale bar, 25 μm. (f) Quantification of MyoD⁺ cells per 100 MFs in TA muscles of Mib1^WT and Mib1^ΔMF mice at indicated ages. n = 5 animals for each group; data are mean ± s.d.; two-sample t-test; ^∗P < 0.05. (g,h) IHC staining (g; scale bar, 25 μm) and quantification (h; n = 5 animals for each group; data are mean ± s.d.; two-sample t-test; N.D. not detected) for Pax7 and MyoG in TA muscles from 4- and 12-week-old Mib1^WT mice. The Pax7⁺/MyoG⁺ cells were observed only in 4-week-old. Statistical source data for h is provided in Supplementary Table 2. (i) Immunoblotting of Myogenin in SCs isolated from hindlimb muscles of 4-week-old Mib1^WT and Mib1^ΔMF mice. Numbers represent the relative signal intensity of the designated antibody measured by densitometry. Unprocessed original scans of blots are shown in Supplementary Fig. 9. (j,k) IHC staining for Pax7 (j), Myogenin (k) and activated Caspase3 in TA muscles from 4-week-old Mib1^WT and Mib1^ΔMF mice. Scale bar, 25 μm. There was no significant increase of apoptotic cells in Mib1^ΔMF mice. (l) mRNA expressions of Pax7, Notch target genes, differentiation markers, and stem cell markers in SCs purified from 4-week-old Mib1^WT and Mib1^ΔMF mice. n = 5 animals for each group; data are mean ± s.d.; two-sample t-test; ^∗P < 0.05.