Fig 2: Gene expression of PGC-1a in skeletal muscles and cells. The expression of PGC-1a was examined in the samples used in Figs 1, 2, 3, 4, 6, and 7A. Each value is presented as the mean ± SE. For FOXO1-Tg experiment, statistical analyses were performed using Student’s two-tailed unpaired t-test (N = 5). **P < 0.01 versus wild-type. For denervation experiment, statistical analyses were performed using Student’s two-tailed unpaired t-test (control: N = 3, denervation: N = 4). ***P < 0.001 versus control. For casting experiment, statistical analyses were performed using Student’s two-tailed unpaired t-test (control: N = 5, casting: N = 4). For fasting experiment, statistical analyses were performed using one-way analysis of variance followed by Tukey’s post hoc test (N = 6). ***P < 0.001 versus fed; ††† P < 0.001 versus fast for 8 h; § P < 0.05 versus fast for 24 h. For FOXO1-KO experiment, statistical analyses were performed using two-way analysis of variance followed by Tukey’s post hoc test (wild-type fed, n = 3; wild-type fasted, n = 4; KO fed, n = 4; KO fasted, n = 4). ***P < 0.001, *P < 0.05. For FOXO1-ER experiment, statistical analyses were performed using Student’s two-tailed unpaired t-test (N = 6). ***P < 0.001 versus control.

Fig 3: Effect of FOXO1 activation on the expression of PGC-1ß in C2C12 cells. (A) Tamoxifen (TAM) was added to FOXO1-ER cells, and 24 h later, mRNA expression was analyzed. Expression levels of FOXO1, cathepsin L, PGC-1ß, and MCAD are shown. Quantitative real-time RT-PCR data from controls were set at 100 arbitrary units. Each value is presented as the mean ± SE (N = 6). Statistical analyses were performed using Student’s two-tailed unpaired t-test. ***P < 0.001, **P < 0.01 versus control. (B) Time course (3, 6, 8, and 24 h) after tamoxifen treatment; microscopic views were observed, and mRNA expression levels were analyzed. Scale bars, 100 µm. Quantitative real-time RT-PCR data from 0 h were set at 100 arbitrary units. Each value is presented as the mean ± SE (N = 4). Statistical analyses were performed using Student’s two-tailed unpaired t-test. ***P < 0.001, **P < 0.01, *P < 0.05 versus control (vehicle).

Fig 4: Gene expression analysis of FOXO1, cathepsin L, PGC-1ß, and MCAD in the skeletal muscles of plaster-casted mice. The expression of FOXO1 and cathepsin L was increased upon unloading using a plaster cast. The expression of PGC-1ß and MCAD was decreased by plaster cast. Quantitative real-time RT-PCR data from control samples were set at 100 arbitrary units. Each value is presented as the mean ± SE (control: N = 5, casting: N = 4). Statistical analyses were performed using Student’s two-tailed unpaired t-test. ***P < 0.001, **P < 0.01, *P < 0.05 versus control.

Fig 5: Gene expression (AU) of (A) Foxo1, (B) Trim63, and (C) Fbxo32 determined by RT qPCR in muscle homogenates from TNF-tg, and TNF-tg11ßKO animals receiving either vehicle or corticosterone (100 µg/mL) in the drinking water for 3 weeks. (D) The p-FOXO1(Thr24)/Total FOXO1 and (E) p-S6(Ser235/236)/Total S6 ratios and (F) representative western blot staining after loading of 20 µg of protein and normalization to Ponceau staining in quadriceps for TNF-tg and TNF-tg11ßKO animals receiving either vehicle or corticosterone (100 µg/mL) in the drinking water for 3 weeks. Gene expression (AU) of (G,H) Mstn and (I,J) Fbxo32 in primary murine muscle cultures from WT and single mutant 11ßKO animals treated with recombinant TNFa and/or dehydrocorticosterone (DHC) determined by RT qPCR. Values are expressed as mean ± SE, n = 6 per group for animal experiments and n = 3 per group for primary culture. Statistical significance was determined using two-way analysis of variance with Tukey post hoc analysis. * p < 0.05, ** p < 0.005, *** p < 0.001. WT, Wild type; 11ßKO, 11ß-HSD1 genetic deletion; VC, Vehicle Control; Cort, Corticosterone; DHC, Dehydrocorticosterone.