Fig 2: SOXF factors modulate satellite cell behavior.(A–E–I) Immunofluorescence of satellite cells transduced with SOXF-encoding retroviruses after 72 hr in culture on isolated adult wild type EDL myofibers. SOXF-FL, construct overexpressing SOXF; SOXF?Ct, altered construct lacking the C-terminus (preserving the HMG DNA binding domain); CTRL, encoding just eGFP. GFP marks transduced cells. Nuclei are counterstained with DAPI (blue). Scale bars, 20 µm. (B–D, F–H, J–L) Quantification of the transduced satellite cells illustrated in (A–E–I) for quiescence (PAX7), activation (MYOD), and proliferation (KI67), compared to CTRL. n = 50 fibers/EDL per condition; =1000 satellite cells/EDL. Data expressed as mean ± s.e.m., statistically analyzed with Student’s unpaired t-test: *, p<0.05; **, p<0.01; ***, p<0.001, compared to CTRL.

Fig 3: Necrostatin Restores Proliferation of Chd4mKO MuSCs and Improves Skeletal Muscle Regeneration in Chd4mKO Mice(A) Immunofluorescence staining for PAX7/pMLKL on TA muscle sections from Chd4mKO and Chd4mKO/Ripk3mKO mice. Scale bar, 25 µm.(B and C) Quantification of PAX7+/pMLKL+ MuSCs (B) undergoing necroptosis and PAX7+ MuSCs (C) in control, Chd4mKO, and Chd4mKO/Ripk3mKO muscles (n = 3 for each group).(D) Fluorescence images of uninjured (left) and injured (right) TA muscles from Chd4mKO/ROSA26YFP, Chd4mKO/Ripk3mKO/ROSA26YFP, and Chd4mKO/ROSA26YFP 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 Chd4mKO/ROSA26YFP TA muscles with and without Nec-1s treatment after CTX-induced muscle injury (n = 3–6 for each group.(G) EthD-III incorporating Chd4mKO 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 Chd4mKO 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.

Fig 4: Activation of NF-?B in MuSCs in the context of repetitive injuries leads to Ku80 dysregulation(A) Isolated MuSCs from 3× injured control and IKK2CAMuSC mice subjected to qRT-PCR for genes associated with telomeric function. Note that XRCC5, which encodes the DNA repair protein, Ku80, is downregulated in IKK2CAMuSC MuSCs. n = 4–7 mice (3–5 months old) per genotype.(B) Representative Ku80 staining (red), VCAM (green), and nuclei (DAPI, blue) in muscle sections from control and IKK2CAMuSC mice after 3× injury.(C) Quantification shows reduced levels of Ku80 in IKK2CAMuSC MuSCs compared to controls. n = 3 samples/group. Mean ± SEM. Statistical analysis was performed using unpaired Student’s t test with Welch’s correction. ***p = 0.001.(D) Representative image of TIFs (telomere-induced foci), where DNA damage (53BP1, green) co-localize with telomere (red) in VCAM-positive cells (white).(E) Quantification shows higher TIFs in IKK2CA-MuSC compared to controls. n = 3 mice (4 months old) per genotype. n > 50 cells per mouse.(F) Left: representative two-photon microscopy images. MuSCs are shown in green, while muscle fibers are visualized in red by second harmonic generation (SHG). Right: quantification of the number of Pax7 (EGFP+) MuSCs shows stem cell exhaustion over time under chronic injuries. n = 3 mice per genotype. All datasets displayed are mean ± SEM. Statistical analyses were performed using unpaired Student’s t test with Welch’s correction. *p = 0.05; ***p = 0.001.

Fig 5: Persistent NF-?B activation in human DMD MuSCs leads to Ku80 dysregulation and predisposes these cells to premature telomere shortening and increased number of TIFs(A) In-cell western representative image of phospho-p65 staining of human MuSCs. MuSCs from three healthy and three DMD patients 10–15 years old were analyzed with five technical replicates each. Green, phospho-p65 staining; red, Draq5 (nuclear) staining.(B) Quantification shows nuclear accumulation of p-p65 in hMuSCs. The total signal intensity in the IR 800 channel (p-p65) was normalized to the total intensity of the Draq5 (nuclear channel) per sample. n = 3 samples/group. n = 6 wells per sample.(C) Higher NF-?B activity in NCAM+ human MuSCs from young DMD patients, as shown by the p-p65 nuclear accumulation compared to age-matched control MuSCs.(D) n = 3 samples/group. Representative Ku80 staining (green), Pax7 (red), and nuclei (DAPI, blue) in human healthy and DMD muscle sections (21–26 years old).(E) Quantification shows reduced levels of Ku80 in dystrophic MuSCs compared to controls. n = 3 samples/group. n > 30 cells per sample.(F) Reduced telomere length in human MuSCs, assessed by MuQ-FISH of telomere staining (insert, representative image). n = 3 samples/group. n > 80 cells per sample.(G) Representative images of TIF assay in human MuSCs.(H) Quantification analysis reveals increased number of TIFs in human MuSCs.n = 3 samples/group. n > 60 cells per sample. Graphed data are displayed as mean ± SEM. Statistical analyses in (B), (E), and (H) were performed using unpaired Student’s t test with Welch’s correction. *p = 0.05; **p = 0.01; ***p = 0.001.