Figure 6.

Necrostatin Restores Proliferation of Chd4^mKO MuSCs and Improves Skeletal Muscle Regeneration in Chd4^mKO Mice

(A) Immunofluorescence staining for PAX7/pMLKL on TA muscle sections from Chd4^mKO and Chd4^mKO/Ripk3^mKO mice. Scale bar, 25 μm.

(B and C) Quantification of PAX7⁺/pMLKL⁺ MuSCs (B) undergoing necroptosis and PAX7⁺ MuSCs (C) in control, Chd4^mKO, and Chd4^mKO/Ripk3^mKO muscles (n = 3 for each group).

(D) Fluorescence images of uninjured (left) and injured (right) TA muscles from Chd4^mKO/ROSA26^YFP, Chd4^mKO/Ripk3^mKO/ROSA26^YFP, and Chd4^mKO/ROSA26^YFP mice treated with Nec-1s 2 weeks after CTX-induced muscle injury (n = 3–5 for each group). Scale bar, 100 μm.

(E) Cross sections of injured TA muscles as in (D) indicating increased formation of YFP⁺ myofibers after Ripk3 inactivation. Scale bar, 25 μm.

(F) Quantification of MuSCs in Chd4^mKO/ROSA26^YFP TA muscles with and without Nec-1s treatment after CTX-induced muscle injury (n = 3–6 for each group.

(G) EthD-III incorporating Chd4^mKO MuSCs after treatment with z-VAD, necrostatin-1, or DMSO (n = 3 for each group); scale bar, 100 μm.

(H) Quantification of EthD-III incorporating Chd4^mKO MuSCs after 100 h of in vitro culture in a field of 1 mm² following treatment with z-VAD, necrostatin-1, or DMSO (n = 3–6 for each group. Statisical analysis: ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.005, two-way ANOVA followed by Bonferroni post-test with alpha = 5%).

All analyses indicated across the experiments were biological replicates unless otherwise stated.