Fig 2: Recombinant ADAMTS1 alleviates muscle atrophy. (A, B) Weights of TA (A) and GA (B) muscles in mice with dexamethasone-induced atrophy. Recombinant ADAMTS1 was injected at a concentration of 1 mg/kg (Low) or 10 mg/kg (High). *P < 0.05, **P < 0.01, ***P < 0.001. (C, D) Histological analysis of the TA muscle. H&E-stained sections (C) and CSA of TA muscle (D). Scale bar: 100 μm. (E) Western blotting analysis of tibialis anterior muscle. (F) Quantification of the mRNA expression of muscle atrophy-related factors Atrogin1 and Murf1. †P < 0.05, ††P < 0.01 compared to the control group, *P < 0.05 compared to the dexamethasone group. Data are presented as the mean ± standard deviation. Statistical significance was tested using one-way ANOVA with Tukey’s post hoc test. Dexa, dexamethasone; rAL, recombinant ADAMTS1 Low (1 mg/kg); rAH, recombinant ADAMTS1 High (10 mg/kg).

Fig 3: Recombinant ADAMTS1 generation and characterization. (A) Schematic representation of recombinant ADAMTS1 mutant. (B, C) Characterization of recombinant ADAMTS1 NM3. Proteins were analyzed under non-reducing and reducing conditions using SDS-PAGE (B) and SE-HPLC (C): (1) purified protein supernatant, (2) purified protein incubated at room temperature for 2 d, (3) concentrated protein, (4) 1M ammonium sulfate added to the cell culture media, and (5) 1M ammonium sulfate added to the solution. (D, E) Characterization of the recombinant ADAMTS1 C198S. Proteins were analyzed under non-reducing and reducing conditions using SDS-PAGE (D) and SE-HPLC (E) of (1) purified protein supernatant and (2) concentrated protein. Pro, pro-domain; MP, metalloproteinase.

Fig 4: Inhibitory effect of recombinant ADAMTS1 on ADAM10-mediated NOTCH1 activation. (A, B) ADAMTS1 knockdown C2C12 cells were treated with recombinant ADAMTS1 protein at a concentration of 100 ng/ml. Differentiated myotubes were visualized using Jenner’s staining (A), and the myotube length was measured (B). Scale bar: 500 μm. *P < 0.05, **P < 0.01, ***P < 0.001. (C) Effect of ADAM10 on muscle cell differentiation. Recombinant ADAM10 protein was added at concentrations of 0.1, 1, 10, and 100 ng/ml. Differentiated myotube length was measured. ***P < 0.001. (D) Western blotting analysis of ADAM10-treated C2C12 cells. Quantification of protein expression using ImageJ software. ***P < 0.001. (E) Immunofluorescence staining of C2C12 cells. Cells were treated with recombinant ADAM10 at 10 ng/ml and recombinant ADAMTS1 at 0 (untreated), 1, 10, 100, and 1000 ng/ml. Scale bar: 100 μm. (F) Western blotting analysis of C2C12 cells treated with recombinant ADAM10 at 10 ng/ml and recombinant ADAMTS1 at 0 (untreated), 1, 10, 100, and 1000 ng/ml. Protein expression was quantified using ImageJ software. †P < 0.05, ††P < 0.01, †††P < 0.001 compared to control group, *P < 0.05, **P < 0.01, ***P < 0.001 compared to recombinant ADAMTS1 untreated group. (G) Quantification of Hes1 mRNA expression. †P < 0.05 compared to control group, **P < 0.01 compared to the untreated group. Data are presented as the mean ± standard deviation. Statistical significance was tested using one-way ANOVA with Tukey’s post hoc test. Con, control; si-ctrl, control siRNA; si-ATS #1, ADAMTS1 siRNA #1; si-ATS #2, ADAMTS1 siRNA #2; rA, recombinant ADAMTS1.