Fig 1: CTRP9 knockdown accelerates senescence and atrophy in differentiated C2C12 myotubes and impairs myogenesis in replicative aging C2C12 myoblasts.A Western blot analysis of p53, p21, p16 and p19 protein expression in C2C12 myotubes transfected with siCTRP9 or siNC; GAPDH serves as the loading control (n = 3). B Immunofluorescence staining of MHC (green) and DAPI (blue) in C2C12 myotubes transfected with siCTRP9 or siNC. The diameter and length of myotubes are measured, and their nuclei are counterstained with DAPI. Representative images are shown (n = 3). Scale bar = 100 μm. C Left: CTRP9 mRNA expression in C2C12 cells at day 0 and day 2 of differentiation. Right: CTRP9 mRNA expression in C2C12 cells treated with PBS or bFGF (10 ng/mL) during differentiation. CTRP9 expression is assessed by qPCR (n = 3). D Western blot analysis of CTRP9 protein expression in siNC and siCTRP9 groups; TUBULIN serves as the loading control (n = 3). E Western blot analysis of MYOD1, p53, p21, p19 and p16 protein expression in siNC and siCTRP9 groups; GAPDH serves as the loading control (n = 3). F Immunofluorescence staining of myosin heavy chain (MHC; green) in the siNC and siCTRP9 groups of C2C12 myoblasts. Nuclei were counterstained with DAPI (blue) (n = 3). Scale bar = 100 μm. Data are presented as mean ± SEM.