Dexamethasone decreases CRTC1 and CRTC2 nuclear localization.

<p>Treatment of L6 myotubes with Dex (100nM, 48hrs) decreased (A) total CRTC1 (p = 0.004 and CRTC2 protein (p = 0.0007). n = 9/treatment from 3 experiments. Representative western blots and corresponding sections of the Ponceau S-stained membranes are shown. Dex decreased (B) nuclear CRTC1 (p = 0.04) and nuclear CRTC2 (p = 0.03) protein and (C) nuclear CnA protein (p = 0.04); no differences in the cytosolic levels of these proteins were detected. Dex increased (D) nuclear SIK1 protein (p = 0.005) with no detectable difference in cytosolic protein. n = 3-7/treatment from 3–5 experiments. (E) Representative western blots for cytosolic and nuclear CRTC1, CRTC2, SIK, and CnA proteins are shown; controls for the nuclear (Histone) and cytosolic (GAPDH) fractions are also shown. (F) IBMX (250uM, 15 min) increased nuclear CRTC2 protein. A representative western blot image and corresponding Ponceau S-stained membrane from a single experiment run on the same gel are shown; the experiment was repeated one other time with similar results. In panels A-D, data are expressed as mean percent of control ± s.d and were analyzed by Student’s <i>t</i>-test. Asterisks indicate a value that is significantly different from control: p<0.05 = *, p<0.01 = ** and p<0.001 = ***.</p

Dexamethasone decreases PGC-1α protein expression and transcription.

<p>(A) Treatment of L6 myotubes with Dex (100nM, 48hrs) decreased PGC-1α protein expression (p = 0.01). n = 3/treatment from 3 experiments. A representative western blot image and corresponding section of the Ponceau S—stained membrane are shown. (B) Dex decreased the mRNAs for PGC-1α (p<0.0001) and its target genes Tfam (p = 0.03) and CytC (p = 0.03); Dex also increased MuRF-1 (p = 0.003) and Atrogin-1/MAFbx mRNAs (p<0.0001). n = 10-14/treatment from 4–5 experiments. (C) In L6 cells transfected with a pPGC-1α-Luc, Dex decreased luciferase activity (p = 0.001). n = 6/treatment from 6 experiments. (D) In separate experiments with cells transfected with a pPGC-1α-ΔCRE-Luc, Dex increased luciferase activity (p = 0.0005). n = 3/treatment from 3 experiments. Data are expressed as mean values ± s.d. and were analyzed by Student’s <i>t</i>-test. Asterisks indicate values that are significantly different from control: p<0.05 = *, p<0.01 = **, p<0.001 = *** and p<0.0001 = ****.</p

Dexamethasone, CRTC2, and atrogene expression.

<p>Two-way ANOVA was used to evaluate whether CRTC2 alters atrogene mRNA responses to Dex. Treatment with Dex increased (A) MuRF-1 mRNA (p<0.0001) and (B) Atrogin-1/MAFbx mRNA (p<0.0001). Overexpression of CRTC2 did not affect MuRF-1 or Atrogin/MAFbx mRNA expression (p≥0.1) or effect the response to Dex treatment (p≥0.1). n = 11-12/treatment from 4 experiments. Data are expressed as mean percent of control ± s.d. Asterisks denote significant effects of Dex compared to respective controls as indicated by <i>post hoc</i> analysis: p<0.01 = **, p<0.001 = *** and p<0.0001 = ****.</p

Overexpression of CRTC proteins increase PGC-1α transcription.

<p>L6 myoblasts were transfected with either pPGC-1α-Luc or pPGC-1α-ΔCRE-Luc. After 24 h, cells were infected with adenoviruses to express GFP, human CRTC1 or human CRTC2 and harvested 48 h later to measure luciferase activity. A two-way ANOVA was used to evaluate the significance of CRTC protein overexpression and the mutation of the CRE site on pPGC-1α-Luc activity. Overexpressing either CRTC1 (A) or CRTC2 (B) increased luciferase activity (CRTC1, p = 0.0001, CRTC2, p = 0.0002). In both sets of experiments, luciferase activity is reduced in cells transfected with pPGC-1α-ΔCRE-Luc (p<0.0001 in each panel). The CRTC1 or CRTC2-induced increase in luciferase activity was prevented by mutation of the CRE site in pPGC-1α-Luc (CRTC1, p = 0.003; CRTC2, p = 0.01). n = 5-6/treatment from 5–6 experiments. Data are expressed as the mean value ± s.d of normalized (i.e., firefly:renilla ratio) luciferase activity. The # indicates that the effects of CRTC1 or CRTC2 on luciferase activity are different at p<0.01 when CRE site is mutated in the PGC-1α promoter. Asterisks denote significant effects of CRTC1 or CRTC2 compared to GFP controls as indicated by <i>post hoc</i> analysis: p<0.001 = ***.</p

Ratio of p-PRAS40 to total PRAS40 protein after 12 hours spontaneous breathing or mechanical ventilation.

<p>Representative western blots are shown below the graph. Values are expressed as mean ± SE change from percent control. Symbols: * significantly different (p<0.05) from all other groups.</p

Ratio of p-AKT to total AKT protein in the diaphragm following 12 hours spontaneous breathing or mechanical ventilation.

<p>Representative western blots are shown below the graph. Values are expressed as (mean ± SE) change from percent control. Symbols: δ significantly different (p<0.05) from SB and CMVSS.</p

Ratio of p-4E-BP1 to total 4E-BP1 protein in the diaphragm following 12 hours of spontaneous breathing or mechanical ventilation.

<p>Representative western blots are shown below the graph. The ratio was determined via analysis of the sum total of all bands in both the phosphorylated and total blots. Values are expressed as mean ± SE change from percent control. Symbols: * significantly different (p<0.05) from all groups.</p

Ratio of p-mTOR to total mTOR protein in the diaphragm following 12 hours spontaneous breathing or mechanical ventilation.

<p>Representative western blots are shown below the graph. Values are expressed as mean ± SE change from percent control. Symbols: * significantly different (p<0.05) from all other groups.</p

Comparison of respiratory work between spontaneously breathing animals (SB) and animals ventilated with PSV.

<p>Key: Ti = inspiratory time; Ttot = time of a breathing cycle; Pneg = inspiratory pressure; TTI—time tension index.</p><p>* indicates p<0.05.</p><p>Comparison of respiratory work between spontaneously breathing animals (SB) and animals ventilated with PSV.</p

Mixed protein fractional synthesis rate (FSR) in the diaphragm following 12 hours of spontaneous breathing or mechanical ventilation.

<p>Values are expressed as percent change per hour (mean ± SE). Note that these 12 hour values represent an average FSR during 6–12 hours of the experiments. Symbols: * significantly different (p<0.05) from all other groups.</p