Fig 1: BGN expression is upregulated, and BGN is secreted throughout differentiation. (A) mRNA expression (normalized to GAPDH) and (B) immunoblotting of BGN and myogenic markers in C2C12 cells during myogenic differentiation and the corresponding quantification. (C) BGN concentration in the culture medium of C2C12 myoblasts and myotubes. (D) BGN mRNA expression and (E) BGN accumulation in the culture medium of C2C12 myotubes after treatment with 2 µM forskolin for the indicated times. The data are presented as the mean ± SD; * p < 0.05, ** p < 0.01, and *** p < 0.001.
Fig 2: BGN decreases with age in the skeletal muscle of humans and mice. (A) Expression levels of biglycan (BGN) and Foxo3 in the quadriceps muscle of young (n = 10) and older (n = 10) human subjects from the GSE175495 dataset. (B,C) Changes in the (B) mRNA (normalized to Hprt) and (C) protein levels of BGN in the gastrocnemius (GA) muscles of young (7-month-old) and older (22-month-old) mice fed a normal chow diet (ND) or ND plus exercise (exe) (n = 6–7 per group). (D) A schematic diagram (created using BioRender.com) showing the Dox-induced aging mouse model. (E) Relative mRNA levels (normalized to GAPDH) of senescence (p16 and p21), atrophy (Foxo3, 4ebp1, and Atrogin), and SASP (IL6, Tgfβ1, and Ccl2)-related genes in the GA muscle of control (PBS) and Dox-injected mice (n = 4 per group). (F) mRNA (normalized to GAPDH) and (G) plasma levels of BGN in control (n = 4) and Dox-treated mice (n = 4). The data are presented as the mean ± SD; * p < 0.05, ** p < 0.01, and *** p < 0.001.
Fig 3: Schematic representation of our proposed mechanism. Graphical diagram (created using BioRender.com) showing the regulatory roles of BGN in aging-associated signaling pathways in skeletal muscle and liver.
Fig 4: BGN improves protein synthesis by activating the AKT/mTOR pathway. (A) Western blot analysis and quantification of total and phosphorylated AKT, mTOR, pS6K, and p4EBP1 in C2C12 myotubes treated with 0.5 µg/mL BGN or PBS (as a negative control). (B) Western blot analysis and quantification of total and phosphorylated S6K and 4EBP1 in C2C12 myotubes treated with BGN (0.5, 1.0, or 1.5 μg/mL) or PBS (as a negative control) for 48 h. (C) Representative immunoblot of puromycin incorporation in C2C12 myotubes and the corresponding quantification. (D) Western blot analysis and quantification of total and phosphorylated S6K, 4EBP1, and S6 in C2C12 myotubes treated with 10 µM Dex with or without 0.5 µg/mL BGN pretreatment for 24 h. The data are presented as the mean ± SD; * p < 0.05, ** p < 0.01, and *** p < 0.001.
Fig 5: BGN mitigates cellular senescence in hepatocytes. (A) Viability of AML-12 hepatocytes treated with 0, 0.2, 0.5, or 1 µM Dox for 24 h. PBS was used as a negative control. (B) Relative mRNA expression of p21 in hepatocytes treated with Dox (normalized to L32 mRNA). (C) Western blot of p21 and γH2AX in hepatocytes treated with Dox and the corresponding quantification. (D) Representative images of senescence-associated β-galactosidase (SA-β-gal) staining of hepatocytes treated with 0.2 µM Dox for 24 h with or without 1 µg/mL BGN pretreatment. Scale bars = 50 µm. (E) Quantification of SA-β-gal-positive cells. (F) Relative mRNA expression of p21, IL6, and TNFα in hepatocytes (normalized to L32 mRNA). (G) Western blot of p21 and Ki67 in hepatocytes and the corresponding quantification. The data are presented as the mean ± SD; * p < 0.05, ** p < 0.01, and *** p < 0.001.
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