Figure 1.
Starving Cells and Glucocorticoid Treatment Induce atrogin-1 Expression and Dephosphorylation of Members of the PI3K/AKT-Signaling Pathway
(A and B) C2C12 myotubes were starved by removal of growth medium and incubated in PBS for 6 hr. Medium was replaced in refed samples for 12 hr.
(A) Effect of starvation on atrogin-1 expression: Northern blots probed for atrogin-1 and GAPDH (middle image) and fold increase in atrogin-1 mRNA, calculated by dividing the atrogin-1 band intensity (atrogin-1/GADPH) with the atrogin-1/GADPH ratio in the control condition (upper image). Both atrogin-1 bands were used in the analysis. Micrographs of representative control, starved, and refed C2C12 myotube cultures (lower image).
(B) Effect of starvation on components of the PI3K/AKT pathway: proteins were extracted from the same samples as in a, and subjected to immunoblot analysis. Quantitation of the ratio of phosphorylated to total protein was determined as above. Data was normalized to a control of 100%.
(C) Effect of 1 μM Dex treatment on atrogin-1 expression.
(D) Effect of Dex on PI3K/AKT pathway components.
(E) Foxo factors are nuclear in starved C2C12 myotubes. Left image: nuclear extracts from control and starved myotubes were immunoblotted with anti-Foxo antibodies. Right image: nuclear extracts were tested for binding to double-stranded 32P-labeled oligonucleotides containing Foxo, CREB, NFκB, and AP1 binding sites by electrophoretic mobility shift assay. Arrow, Foxo–oligonucleotide complexes. Asterix, nonspecific band.