Figure 4: Acute inhibition of FoxOs phenocopies the conditional FoxO1,3,4 knockout.
(a) Frequency histograms of gastrocnemius muscles showing the distribution of cross-sectional areas (μm2) of inducible muscle-specific FoxO1,3,4 mice after tamoxifen-dependent deletion of FoxO1,3,4 genes (FoxO1,3,4f/f: black bars and FoxO1,3,4−/−: magenta bars) in fed (upper panel) and fasted (lower panel) conditions, n=3, each group. (b) Force measurements preformed in vivo on gastrocnemius muscle showed that acute inhibition of FoxOs in adulthood prevents force drop during fasting. n=6 muscles in each group. Freq: Frequency. (c) Left, immunoblotting analyses of gastrocnemius homogenates after acute deletion of FoxO1,3,4−/− and controls. Right, quantification of LC3 lipidation. Data are representative of three independent experiments. (d) Autophagy flux is not increased in FoxO-deficient TA muscles. Inhibition of autophagy–lysosome fusion by colchicine treatment induces accumulation of LC3II band in starved control but not in starved FoxO1,3,4−/− muscles. Left, immunoblotting analyses of gastrocnemius homogenates. Right, quantification of LC3 lipidation. (e,f) ChIP quantitative RT–PCR shows the recruitment of FoxO3 and FoxO1 on the promoters of selected atrophy-related genes. ChIP assays were performed in starved control and FoxO1,3,4−/− TA muscles. IgG was used as the reference. n=3 for each group. Data are shown as mean±s.e.m. Error bars indicate s.e.m. *P<0.05, **P<0.01 (Student’s t-test). S1: FoxO binding site 1; S2: FoxO binding site 2.
