Fig. 3: Calcr-cKO MuSC proliferation in non-increased-loading muscles, EDL and TA, following exercise. | Nature Communications

Fig. 3: Calcr-cKO MuSC proliferation in non-increased-loading muscles, EDL and TA, following exercise.

From: Calcitonin receptor downregulation and exercise-conditioned blood enable systemic muscle stem cell proliferation

Fig. 3

A Experimental scheme for analyzing MuSC behavior in EDL and TA in exercised control (+Ex) (Cont; Pax7CreERT2::Rosayfp/yfp, n = 3), exercised C-cKO (+Ex) (Pax7CreERT2::Calcrflox/flox::Rosayfp/yfp, n = 5), or non-exercised C-cKO (−Ex) (n = 3) using immunohistochemistry (IHC). B Count of wheel rotations per night of Cont (n = 3) or C-cKO (n = 5) mice for 2 weeks-exercise with mean ± s.d.; unpaired two-tailed Student’s t test. C Verification of EdU+ myonuclei (white) located beneath dystrophin (green) in EDL of C-cKO (−Ex) and (+Ex). Arrowheads indicate EdU+ myonuclei in EDL of C-cKO (+Ex) mice. Scale bar, 50 µm. D Number of EdU+ myonuclei in EDL or TA in Cont (+Ex, n = 3), C-cKO (−Ex, n = 3), or C-cKO (+Ex, n = 5) mice. E Immunostaining for LNα2 (green), M-cadherin (red), and EdU (white) in EDL from C-cKO (+Ex) mice. Scale bar, 10 µm. F Number of EdU+M-cadherin+ MuSCs in EDL or TA in Cont (+Ex, n = 3), C-cKO (−Ex, n = 3), or C-cKO (+Ex, n = 5) mice. G Experimental scheme for analyzing MuSC number on isolated single myofibers (SMF) from EDL of exercised Cont (n = 5), C-cKO (n = 8), C-cKO/PKA (Pax7CreERT2::Calcrflox/flox::PKA-tg::Rosayfp/yfp, n = 4), or C/Y-cdKO (Pax7CreERT2::Calcrflox/flox::Yap1flox/flox::Rosayfp/yfp, n = 7) (+Ex) after 2 weeks of exercise. SMF of non-exercised Cont (n = 4), C-cKO (n = 7), C-cKO/PKA (n = 3), and C/Y-cdKO (n = 4) (−Ex) mice were also analyzed. H Count of wheel rotations per night of Cont, C-cKO, C-cKO/PKA, or C/Y-cdKO for 2 weeks of exercise. I Detection of YFP+Pax7+ MuSCs on isolated myofibers from C-cKO (−Ex) or (+Ex). Arrowheads indicate YFP+Pax7+ cells. Scale bar, 50 µm. J Number of MuSCs per single myofiber from EDL of Cont (−Ex, n = 4), Cont (+Ex, n = 5), C-cKO (−Ex, n = 7), or C-cKO (+Ex, n = 8) mice. K Frequency of myofiber harboring >10 MuSCs on one myofiber in (J). L Number of MuSCs per single myofiber from C-cKO/PKA (−Ex, n = 3), C-cKO/PKA (+Ex, n = 4), C/Y-cdKO (−Ex, n = 4), or C/Y-cdKO (+Ex, n = 7) mice. M Percentage of myofiber harboring >10 MuSCs on one myofiber in (L). The graphs show individual data with mean ± s.d.; one-way ANOVA with Tukey’s multiple comparison test (D, F, H, J–M). The reproducibility of the data was confirmed using more than three biological replicates (C, E, I).

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