Fig 2: LPS-induced cardiac dysfunction could be improved by RIPK3 deletion (n = 6/group). (A–D) Cardiac function was measured via echocardiography. (E–J) Cardiomyocyte mechanical parameters were monitored using a VEVO 2,100 high-resolution imaging system. Cardiomyocytes were isolated from LPS-treated mice and melatonin-treated mice. Then, the resting cell length, peak shortening, time-to-90% re-lengthening (TR90), time-to- shortening (TPS), the maximal velocity of shortening (+dL/dt) and the maximal velocity of re-lengthening (-dL/dt) were determined. *p < 0.05 vs. WT group. #p < 0.05 vs. WT + LPS group.

Fig 3: MG53 inhibits necroptosis in cardiac I/R injury via regulating RIPK1. (A) Western blot analysis of RIPK1, RIPK3, and caspase-8 (necroptosis markers), cleaved caspase-3 (apoptosis marker), and LC-3 II (autophagy marker) in infarct (wt I/R and KO I/R) and non-infarct (wt Non and KO Non) areas of wt and mg53–/– hearts. n = 4 per group. (C) The same set of markers was analyzed by western blot in infarct (wt I/R and tPA I/R) and non-infarct (wt Non and tPA Non) areas of wt and tPA-MG53 hearts. n = 4 per group. (B,D) Western blot results were quantified by ImageJ. (E) Representative western blot image of the expression of cell death pathway markers in hiPSC-derived cardiomyocyte at indicated time points of H/R stress with or without rhMG53 protein. n = 3 independent experiments. Diff 10 days: cells at basal condition (10 days after differentiation), Hypoxia 12 h: cells in hypoxia for 12 h, H/R 1 h (2 h, 4 h): cells with reperfusion for 1 h (2 h, 4 h).

Fig 4: Necroptosis and Apoptosis of MuSCs Ameliorate Muscle Function in Chronic Muscle Disease(A) Schematic representation of the tamoxifen regimen.(B) Immunofluorescence staining of TA muscle cross-sections from mdx and mdx/Ripk3mKO mice 2 weeks after tamoxifen treatment using antibodies against PAX7 and cleaved CASP3 (n = 3 for each group). Scale bar. 25 μm.(C) Quantification of apoptotic CASP3+/PAX7+MuSCs in TA muscle cross-sections.(D) Quantification of total PAX7+ MuSCs in TA muscle cross-sections.(E) Schematic representation of the tamoxifen regimen, z-VAD inhibitor treatment, muscle grip strength measurement, and time points of TA muscle collection.(F–I) Immunofluorescence staining of TA muscle cross-sections from mdx and mdx/Ripk3mKO mice for PAX7 and cleaved CASP3 (F) and RIPK3 (H) 2 weeks after tamoxifen treatment and DMSO or z-VAD inhibitor treatment (n = 3 or 4 for each group) and quantified in (G) and (I), respectively. Scale bar, 25 μm.(J) H&E staining of TA muscles sections from mdx and mdx/Ripk3mKO mice treated with DMSO or z-VAD inhibitor (n = 3 for each group). Scale bar, 100 μm.(K) Quantification of muscle grip strength of mdx and mdx/Ripk3mKO mice treated with DMSO or z-VAD inhibitor (n = 3 or 4 for each group).Statistical 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 5: miR‐183‐5p targets RIPK3 and PDCD4. A, Sequence analysis of miR‐183‐5p binding with the 3′‐UTR of PDCD4. B, Luciferase report assay showing the reduction of luciferase activity in NSC‐34 cells with wild‐type PDCD4, but not the mutant. C and D, Western blots showing the protein levels of PDCD4 in NSC‐34 cells transfected with miR‐183‐5p mimics, inhibitors or scramble sequence (100 nmol/L, respectively). E, Sequence analysis of miR‐183‐5p binding with the 3′‐UTR of RIPK3. F, Luciferase report assay showing the reduction of luciferase activity in NSC‐34 cells with wild‐type RIPK3, but not the mutant. G and H, Western blots showing the protein levels of RIPK3 in NSC‐34 cells transfected with miR‐183‐5p mimics, inhibitors or scramble sequence (100 nmol/L, respectively). Results were averages of four independent experiments. Data represent mean ± SEM. *P < .05, **P < .01 and ***P < .001, n = 3