Fig 2: TWEAK signaling promotes muscle cell fusion during muscle regeneration.A Experimental scheme. The left tibialis anterior (TA) of wild-type mice was injured with an intramuscular injection of 30 μl of 10 μM cardiotoxin to induce focal muscle regeneration. Mice received daily intraperitoneal injections of vehicle (control), 5 μg/kg TWEAK, or 25 μg/kg TWEAK from 3 to 6 dpi. Injured (left) and uninjured (right) TA muscles were collected at 7 and 14 dpi. B Representative cross-sections of TA muscle at 7 dpi stained with laminin (green) and DAPI (blue). Scale bar, 200um. Quantification of (C) minimum Feret diameter of regenerated muscle fibers, D fiber size distribution, and E percentage of fibers containing two or more centrally located nuclei indicative of regeneration from mice treated as in (A) (n = 8 mice). F Representative cross-sections of TA muscle at 14 dpi stained with laminin (green) and DAPI (blue). Scale bar, 200 μm. Quantification of (G) minimum Feret diameter of regenerated muscle fibers, H fiber size distribution, and I percentage of fibers containing two or more centrally located nuclei from mice treated as in (A) (n = 8 mice). Data are presented as mean ± SEM. A one-way ANOVA followed by an uncorrected Fisher’s LSD test was used to compare TWEAK-treated mice to control mice. * and ** denote a significant difference of 0.05, and 0.01, respectively.

Fig 3: TWEAK upregulates genes involved in cellular movement and cell-to-cell signaling and interaction.Global RNA-sequencing was performed on C2C12 myotubes treated with 10 ng/ml TWEAK for 12 h and compared to untreated controls. A Volcano plot of differentially expressed gene (DEGs) among all annotated Ensembl genes. Genes with ≥1.5-fold change and P < 0.05 are shown in red (upregulated) and blue (downregulated); non-significant genes are shown in gray (n = 3). B Ingenuity Pathway Analysis (IPA) of DEGs highlighting top molecular and cellular functions and ranked by z-score. C Venn diagram showing overlap between IPA-identified genes in the “cellular movement” and “cell-to-cell interaction” categories and known cell fusion-related genes (GO: 0140253). D Heatmap of overlapping cell fusion genes differentially expressed in TWEAK-treated myotubes. E RT-qPCR validation of selected targets in C2C12 myotubes treated with 10 ng/ml TWEAK for 6 h (n = 4). F RT-qPCR analysis of Tnfsf12, Cxcl10 and Mymx gene expression in CTX-injured TA muscle (7dpi) from wild-type and TWEAK KO mice (n = 5). G Representative western blot showing Myomixer expression in control and TWEAK-treated C2C12 myotubes at 12 and 24 h post-treatment. H Quantification of Myomixer protein levels from (F) (n = 3). I C2C12 myoblasts co-transfected with non-targeting siRNA (NTi) or siTWEAK and pCMV6 or pCMV6-Mymx plasmid, were differentiated for 4 days. Representative images show MyHC (green) and DAPI (blue) staining. Scale bar, 200 μm. J Quantification of fusion index in cells treated as in (H) (n = 3). RT-qPCR analysis of (K) Tnfsf12 and L Mymx from cells treated as in (H) but collected 3 days in differentiation media. Data are presented as mean ± SEM. Statistical comparisons were performed using unpaired Student’s t test for control vs. TWEAK-treated samples and wild-type and TWEAK KO samples. Two-way ANOVA followed by uncorrected Fisher’s LSD was performed for siTWEAK/Mymx comparisons *, **, *** and **** denote a significant difference of p < 0.05, 0.01, 0.001 and 0.0001, respectively.

Fig 4: Mymx and Cxcl10 are direct transcriptional targets of TWEAK-induced alternative NF-κB signaling.A TransAm assay measuring NF-κB DNA-binding activity in C2C12 myotubes 6 h after treatment with 10 ng/ml TWEAK (n = 4). B Representative images of regenerating TA muscle from control and 5 μg/kg TWEAK-treated mice stained for RelB (pink) and laminin (white). Nuclei are counterstained with DAPI (blue). Scale bar, 50 μm. C Quantification of nuclear RelB in regenerating fibers of mice treated as in (B) (n ≥ 3). RT-qPCR analysis of (D) Nfkb2 and E Relb expression in C2C12 myoblasts transfected with non-targeting siRNA (NTi) or siRNA targeting Nfkb2 (siNfkb2) or Relb (siRelb), differentiated for 3 days, and then treated with 10 ng/mlTWEAK for 12 h (n = 3). F Representative immunofluorescence images of myotubes from cultures treated as in (D, E) and exposed to TWEAK for 24 h. MyHC (green) and DAPI (blue) staining. Scale bar, 200 μm. Quantification of (G) fusion index and H average number of nuclei per myotube from images shown in (D) (n = 3). RT-qPCR analysis of (I) Mymx and J Cxcl10 expression in cells treated as in (D, E) after 24 h of TWEAK exposure. K Schematic of Mymx (top) and Cxcl10 (bottom) regulatory regions showing position of conserved NF-κB DNA-binding motifs relative to the transcription start sites. Chromatin immunoprecipitation (ChIP) analysis of p65 and RelB occupancy on the (L) Mymx promoter or M Cxcl10 promoter in C2C12 myotubes treated with 10 ng/ml TWEAK for 6 h, compared to untreated controls. RT-qPCR values are shown as a percentage of input (n = 3). Data are presented as mean ± SEM. Statistical significance was assessed using two-way ANOVA followed by uncorrected Fisher’s LSD test. *, **, ***, and **** denote a significant difference of p < 0.05, 0.01, 0.001, and 0.0001, respectively.

Fig 5: TWEAK is required for efficient myoblast fusion.A Endogenous TWEAK levels measured by ELISA in culture supernatants of C2C12 myoblasts collected daily during differentiation. Day 0 corresponds to proliferating myoblasts (n = 5). B Western analysis of Fn14 and myogenic factors during C2C12 differentiation. C C2C12 myoblasts were transfected with non-targeting (NTi; black bars) or siRNA targeting murine TWEAK (siTWEAK, blue bars) and differentiated for 4 days. Representative images of myotubes stained with MyHC (green) and counterstained with DAPI (blue). Scale bar, 200um. Quantification of (D) differentiation index, E fusion index, F average number of nuclei per myotube, and (G) the percentage of unfused MyHC+ cells for cells transfected and differentiated as in (A) (n = 6). H RT-qPCR analysis of Tnfsf12 (TWEAK-encoding mRNA) and myogenic markers in NTi- or siTWEAK-transfected cells differentiated for 3 days (n = 4). I TWEAK protein levels in TA muscles at 0, 1, 3, 5, 7, 14, and 28 dpi following cardiotoxin-induced muscle damage, assessed by ELISA (n = 4). J The left TA of wild-type mice was injured with injection of 30 μl of 10 μM cardiotoxin. Mice received daily intraperitoneal injections of vehicle or 1 mg/kg anti-TWEAK neutralizing antibody from 3 to 6 dpi. TA muscles were collected at 14 dpi. Representative laminin (green) and DAPI (blue) stained cross-sections of regenerating muscle at 14 dpi. Scale bar, 200 μm. Quantification of (K) minimum Feret diameter of regenerated muscle fibers and L percentage of fibers containing two or more centrally located nuclei from mice treated as in (J) (n ≥ 5 mice). M TA muscles of wild-type and TWEAK knockout mice were injured with cardiotoxin. Two days post-injury, mice received a single intraperitoneal injection of EdU to label proliferating cells, and TA muscles were harvested 12 days later. Representative images of muscle sections stained for laminin (green), EdU (red), and DAPI (blue). Scale bar, 100 μm. Quantification of (N) minimum Feret diameter of regenerating fibers and O the number of centrally located EdU-positive nuclei per fiber (n ≥ 10 mice). Data are presented as mean ± SEM. Statistical analysis was performed using repeated-measures ANOVA with uncorrected Fisher’s LSD test for time-course comparisons, and unpaired Student’s t-tests for siRNA and in vivo antibody experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.