Fig. 7

FoxO3 and AMPK directly associate to catabolic genes promoters in skeletal muscles of cachectic mice. a Left panel: scheme of the bioinformatics analysis used to generate the average profile of BRD4 ChIP-seq tag counts around ±2 kb of DHSs neighboring a subset of FoxO-regulated genes in control, Cachexia, and Cachexia/JQ1 + mice (right panel). b FoxO3 ChIP qPCR at promoters of muscle catabolic genes and beta globin gene as a negative control. IgG was used as a reference; n = 3. Data represent means ± SEM. Statistical analysis was performed by using one-way ANOVA followed by Tukey’s post hoc test. *p < 0.05; “a” indicates statistical significance compared to control; “b” indicates statistical significance compared to C26 + (−)-JQ1 20 mg/kg/day. c p-AMPK(Thr172) ChIP qPCR at promoters of muscle catabolic genes. Beta globin gene is employed as a negative control. IgG was used as a reference. Animals per group: n = 3. Data represent means ± SEM. Statistical analysis was performed by using one-way ANOVA followed by Tukey’s post hoc test. *p < 0.05; “a” indicates statistical significance compared to control. d C2C12 were transduced with FoxO3-WT or FoxO3-6A retroviruses and allowed to differentiate for 4 days. Myotubes were then maintained in a DM medium with 25 mM glucose or starved in a medium with 0.5 mM glucose and 0.1% horse serum, for 24 h. Immunofluorescence was performed with ant-MyHC antibody (MF20). Scale bar: 50 μm. Right panel: mean diameter of FoxO3-WT and FoxO3-6A myotubes is shown. Data represent means ± SEM. Statistical analysis was performed by using one-way ANOVA followed by Tukey’s post hoc test. ***p < 0.001 vs. FoxO3-WT in DM. e Total RNA from myotubes treated as in d was extracted, and MuRF1 and GABARAPL1 transcript levels were measured by quantitative RT-PCR. Data represent means ± SD. Statistical analysis was performed by using one-way ANOVA followed by Tukey’s post hoc test. *p < 0.05 vs. FoxO3-WT in DM