Figure 3.

Foxo3 Induces atrogin-1 Expression and Causes Reduction in Myotube Size

(A) Foxo3 induces atrogin-1 expression. Myotubes were infected with adenoviral vectors for FOXO3A and c.a. FOXO3A in the absence or presence of IGF-1 (10 ng/ml) and after 48 hr, atrogin-1 mRNA levels were assayed and depicted as in Figure 1. Overexpressed proteins were detected by anti-HA immunoblot.

(B) The atrogin-1 promoter is activated by Foxo3. Myoblasts were transfected with the atrogin-1 reporter constructs 1.0AT1 or 3.5AT1, differentiated for four days, and then infected with FOXO3A or with a control (GFP) vector for 24 hr. Extracts were assayed for Firefly and Renilla luciferase activity. Firefly/Renilla activity was normalized to 1.0 in the control (GFP) infection.

(C) Fluorescence microscopy of myotube cultures overexpressing Foxo3. Myotube cultures were infected with control adenovirus (GFP) or c.a. FOXO3A and photographed 48 hr after infection. Mean myotube diameter from each culture was quantified from three independent experiments.

(D) D.n. Foxo3 inhibits dex-induced atrogin-1 expression and reduction in myotube diameter. Myotubes were infected with adenoviral vectors expressing d.n. FOXO3A, c.a. FOXO3A, or GFP, and incubated in the absence or presence of 1 μM Dex for 24 hr. Overexpressed d.n. FOXO3A was detected by anti-HA immunoblot. Left image: Northern analysis of atrogin-1 expression. Middle image: fluorescence microscopy of myotube cultures after 48 hr. Right image: quantification of mean myotube diameters in the presence of Dex and Foxo expression.