Fig 1: TLR13 knockdown alleviates chronic kidney disease (CKD)-induced loss of muscle mass. (A) Tibialis anterior (TA) muscles of normal C57B6J mice were transfected by intramuscular injection of TLR13 siRNA. Cross-sections of the tibialis anterior muscle from CKD versus control mice were immunostained with TLR13 (red) (scale bar = 20 µm). (B) Tibialis anterior (TA) muscles of control (CTL) and CKD mice were transfected with scrambled control siRNA (siCTL) or siRNA for TLR13 (siTLR13). The expression of TLR13 (mRNA and protein) was examined by immunoblots at 12 days after electroporation (mean ± SEM; n = 6). (C, D) The cross-sectional area of myofibers in TA muscles of CTL and CKD mice following electroporation with siCTL and siTLR13 is shown. The distribution of myofiber sizes in muscles from sham-operated mice treated with siCTL and siTLR13 was comparable (upper panel, data were obtained from three animals in each group). In the muscles of CKD mice treated with siTLR13, the distribution of myofiber sizes was shifted toward the right. Data were obtained from six animals in each group (scale bar = 20 µm). (E) The expression of atrogin-1 and MuRF1 mRNAs was accessed by RT-PCR. TLR13 knockdown suppressed atrogin-1 and MuRF1 expression in the muscle of CKD mice (mean ± SEM; n = 6, *p < 0.05 vs. sham group, # p < 0.05 vs. NCsiTLR13+CKD)
Fig 2: Impact of FAC treatment on myotube size and signalling pathway involved in muscle mass regulation and mitochondrial function. Differentiated C2C12 myotubes were exposed to various concentrations of FAC (10 and 50 µM) in serum-free medium for 72 h. (A) Representative morphology of myotubes at 72 h after vehicle or FAC treatment. (B) Quantitative analysis of myotube diameter. (C) Protein expression or activation of key proteins involved in proteolysis, protein synthesis, and mitochondrial function in response to FAC 10 and 50 µM. (D) Representative blotting images used for the quantification shown in (B). Values are the mean ± SD. The experiments were performed on three independent replicates of three dependent samples for each condition. Significance was checked using Kruskal–Wallis and Dunn's multiple comparisons. Significant differences between conditions (** P < 0.01). 4EBP1, eukaryotic translation initiation factor 4E-binding protein 1; Akt, protein kinase B; COXIV, cytochrome c oxidase IV; Cyt c, cytochrome c; HSC70, heat shock cognate 71 kDa protein; Murf1, muscle RING-finger protein-1; MHC, myosin heavy chain; Nf-?B, nuclear factor-?B; Ub Prot, polyubiquitinated proteins.
Fig 3: Effects of eldecalcitol on muscle atrophy markers in skeletal muscle of ORX mice (A-E) The mRNA level and the protein expression level of muscle atrophy makers Atrogin-1 and MuRF1. *p < 0.05 versus sham; **p < 0.01 versus sham; # p < 0.05 versus ORX; ## p < 0.01 versus ORX. Data are expressed as mean ± SEM, n = 3. Abbreviations: ELD, eldecalcitol; ORX, orchiectomy; SEM, standard error of mean.
Fig 4: Effect of CMH supplementation on UP pathway related genes expression of myotubes under CORT condition. The mRNA expression of FoXO1 (A), MuRF1 (B), Atrogin1 (C), and MSTN (D) were determined and the protein expression of MuRF1 (E, F), Atrogin1 (E, G), and MSTN (E, H) were also evaluated. Data are presented as the means ± SD (n = 6). *P < 0.05, **P < 0.01, and ***P < 0.001. Abbreviations: CMH, creatine monohydrate; CORT, corticosterone.
Fig 5: miR-672-5p Treatment Mitigates Ovariectomy-Induced Sarcopenia(A) Body weight, (B) lean mass, and (C) serum CK levels. Data are mean ± SE. *p < 0.05 and **p < 0.01 compared with the PBS-treated group, or as shown ˆp < 0.05, ˆˆp < 0.01, and ˆˆˆp < 0.001 (n = 6 mice per group). (D) Representative photomicrographs of H&E-stained sections of gastrocnemius muscle from the indicated groups (n = 3, magnification 40×). (E) Cross-sectional area (CSA) and (F) Feret’s diameter. Data are mean ± SE. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the PBS-treated group, or as shown ˆˆˆp < 0.001. (G and H) Immunostaining for laminin (G) and immunostaining for Atrogin-1 (H) (n = 3, magnification 40×). mRNA expression (qPCR, in triplicate) of MyoD (I), Atrogin-1 (J), and MuRF-1 (K) and protein expression (L) (western blotting) of muscle differentiation marker; MyoD (MA1-41017-Thermo Scientific; 1:1,000), catabolic markers, atrogin-1 (ab74023-Abcam; 1:1,000), and muscle ring-finger protein-1 (MuRF-1, ab183094-Abcam; 1:1,000) from gastrocnemius muscles of indicated groups. ß-actin (sc-47778 Santa Cruz Biotechnology; 1:500) was taken as a loading control. Secondary antibodies (either anti-rabbit or anti-mouse; 1:10,000) were HRP conjugated (Sigma-Aldrich). Data are mean ± SE. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the PBS-treated group.
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