1. Volume 14, Issue 3, Pages 395-403 (May 2004)
The IGF-1/PI3K/Akt Pathway Prevents Expression of Muscle Atrophy-Induced Ubiquitin Ligases by Inhibiting FOXO Transcription Factors 
Trevor N. Stitt, Doreen Drujan, Brian A. Clarke, Frank Panaro, Yekatarina Timofeyva, William O. Kline, Michael Gonzalez, George D. Yancopoulos, David J. Glass 
Molecular Cell 
Volume 14, Issue 3, Pages (May 2004)
DOI: /S (04)

2. Figure 1
Dexamethasone Treatment Induces Atrophic Responses in C2C12 Myotubes, Including the Upregulation of Atrophy Markers MAFbx and MuRF1
Myotubes at 3 days of differentiation were treated with dexamethasone for 24 hr and then assessed for morphological changes, changes in protein content, and expression of MAFbx and MuRF1 RNA by Northern analysis.
(A) Phenotypic changes in C2C12 myotubes following dexamethasone treatment. Cultures were treated with 10 μM dexamethasone (DEX) or with vehicle (CON) and fixed after 24 hr (scale bar, 50 μm).
(B) Measurements of average myotube diameter after 24 hr of dexamethasone treatment at the indicated concentrations. Results are presented as the mean (n ≥ 50 myotubes per condition) ± SEM. Asterisk indicates significant difference versus control (p < 0.05).
(C) Quantification of changes in total culture protein levels in response to dexamethasone. Protein concentrations in the nonionic detergent-soluble fraction (open bars) and the remaining detergent-insoluble fraction (after solubilization in boiling 2% SDS; filled bars) were determined after 24 hr treatment at the indicated concentrations and expressed as total protein per 10 cm plate (n = 3 plates per condition) ± SEM. Asterisk indicates significant difference versus control (p < 0.05).
(D) Northern blots showing the effect of 24 hr treatment with increasing concentrations of dexamethasone on MAFbx and MuRF1 expression.
Molecular Cell  , DOI: ( /S (04) )

3. Figure 2
IGF-1 Causes Myotube Hypertrophy and Blocks Dexamethasone-Induced Atrophy
(A) Photomicrographs of myotube cultures that were treated with vehicle alone (upper left panel), 10 ng/ml IGF-1 (lower left), 10 μM dexamethasone (DEX; upper right), or a combination of DEX (10 μM) and IGF-1 (10 ng/ml) (lower right panel). Cultures were fixed after 24 hr; scale bar, 50 μm.
(B) Quantification of average myotube diameter after 24 hr of either no treatment, 10 μM DEX, 10 ng/ml IGF-1, or the combination of 10 μM DEX and 10 ng/ml IGF-1. Data are plotted as the mean (n ≥ 50 myotubes per condition) ± SEM. Asterisk indicates significant difference versus control (p < 0.05); “†” indicates significant difference versus DEX (p < 0.05).
(C) Northern blots showing the opposing effect of IGF-1 (10 ng/ml) on the upregulation of MAFbx and MuRF1 expression by increasing amounts of DEX.
Molecular Cell  , DOI: ( /S (04) )

4. Figure 3
Expression of Constitutively Active PI3K or Constitutively Active Akt Blocks DEX-Induced Upregulation of Atrophy Markers
(A) C2C12 myotubes stably expressing constitutively active PI3K (caPI3K) or control culture cells expressing EGFP (GFP) were differentiated for 3 days, treated with the indicated amounts of DEX for 24 hr, and assessed for MAFbx and MuRF1 expression via Northern blot.
(B) C2C12 myotubes stably expressing constitutively active Akt:GFP fusion (caAkt-GFP) or control cultures (GFP) were treated and probed for MAFbx and MuRF1 expression as in (A).
Molecular Cell  , DOI: ( /S (04) )

5. Figure 4
IGF-1 Treatment Results in Phosphorylation of FOXO Transcription Factors, which Is Necessary for Blockade of Glucocorticoid-Induced Transcriptional Changes
(A) C2C12 myotubes with or without 24hr DEX treatment received IGF-1 (10 ng/ml) for the indicated times. Samples were analyzed by immunoblot with a phosphospecific antibody that recognizes FOXO1 (Thr24) and FOXO3 (Thr32) (top panel) or with an antibody directed against total FOXO3 (bottom panel).
(B) C2C12 myotubes at day 2 of differentiation were infected with adenoviruses expressing myc-tagged FOXO1 (wtFOXO1; left panels) or constitutively active FOXO1 with triple mutations of the Akt phosphorylation sites (caFOXO1; right panels). Cells were serum starved for 20 hr beginning at day 3 and then incubated for 3 hr without (−LY) or with (+LY) the PI3K inhibitor LY FOXO1 localization was detected by immunostaining with anti-myc 9E10 antibody (scale bar, 50 μm).
(C) Transduction of myotubes with a mutant FOXO1 that is resistant to Akt-dependent inactivation prevents IGF-1-mediated effects on atrophy marker expression. Myotubes were infected with adenoviruses encoding wtFOXO1 or caFOXO1 as in (B); an adenovirus expressing EGFP was used as a control (GFP). Transduced cultures were treated at day 3 for a further 24 hr with either no treatment, 10 μM DEX, 10 ng/ml IGF-1, or the combination of 10 μM DEX and 10 ng/ml IGF-1 and processed for Northern blots with MAFbx and MuRF1 probes. Note that in the triple mutant caFOXO1-transduced cultures IGF-1 fails to block the DEX-induced increases in MAFbx and MuRF1 expression.
Molecular Cell  , DOI: ( /S (04) )

6. Figure 5
Intramuscular Administration of IGF-1 Induces Akt Phosphorylation, Inhibits Denervation-Induced Muscle Loss, and Blocks the Increased Expression of MAFbx and MuRF1
(A) IGF-1 induces IGF-1 receptor and Akt phosphorylation. IGF-1 (100 μg) was injected intramuscularly into mouse gastrocnemius complex and the muscles harvested at 0, 15, 30, and 60 min postinjection. Muscle protein was subjected to SDS-PAGE and immunoblotted with a phosphospecific antibody that recognizes phosphotyrosines 1135 and 1136 in the activation domain of the IGF-1 receptor (top panel), with a phosphospecific antibody to Ser473 in Akt (middle panel), or with an antibody to Akt (bottom panel).
(B) Intramuscular injection of IGF-1 partially rescues denervation-induced muscle loss. Denervation atrophy was induced in the mouse gastrocnemius complex unilaterally by sciatic nerve transection and allowed to proceed for 10 days. Muscles received four injections of either saline vehicle or 200 μg IGF-1 protein on days 3, 5, 7, and 9; on day 10 the muscles were collected and weighed. Data is expressed as mean percent loss of wet weight versus weight of the muscle on the unperturbed side (n = 5). Asterisk indicates significant difference versus saline-injected control (p < 0.05).
(C) Northern analysis of MAFbx and MuRF1 transcript levels in control (Nonperturbed) and denervated (Denerv) gastrocnemius muscles shown in (B) demonstrating that the denervation-induced upregulation is blocked by intramuscular administration of IGF-1.
Molecular Cell  , DOI: ( /S (04) )