Rapamycin and Torin1 effects on neurite outgrowth in presence of HMB.

<p>Cells were pre-treated with rapamycin 20nM or Torin1 10 nM and then treated with 25 μM HMB for 48 h. The inhibitor was maintained during the experiment. Results represent means ± SEM (n = 8). * p<0.05 versus control cells. # p<0.05 versus HMB treated cells.</p

HMB activates mTOR and promotes protein synthesis in Neuro2a cells.

<p><b>(A)</b> Neuro2a cells were pre-treated with rapamycin 20nM or Torin1 10 nM and then were treated with 25 μM HMB for 30 min. Western blot analysis was performed using specific antibodies against phospho- and total-antibodies mTOR. <b>(B)</b> Protein synthesis was measured in Neuro2a cells incubated with 25 μM HMB for 2 hours in the absence (n = 10) or presence of inhibitors of PI3K/Akt (LY294002 20μM), ERK1/2 (PD98059 10μM) or mTOR (rapamycin 20nM). Pretreatment of inhibitors occurred as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135614#pone.0135614.g002" target="_blank">Fig 2</a>. Results represent means ± SEM (n = 4). * p<0.05 versus control cells. # p<0.05 versus HMB treated cells.</p

HMB induced neurite outgrowth is mediated by PI3K/Akt and ERK1/2 signaling pathways in Neuro2a cells.

<p>Neuro2a cells were treated with 25 μM HMB for 30 min. Western blot analysis was performed using specific antibodies against phospho- and total-antibodies against Akt <b>(A)</b> and ERK1/2 <b>(B)</b>. <b>(C)</b> Neuro2a cells were pre-treated with LY294002 20μM or PD98059 10μM and then treated with 25 μM HMB for 48 h. Inhibitors were maintained during the experiment. Cell morphology was measured as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135614#pone.0135614.g001" target="_blank">Fig 1</a>. Results represent means ± SEM (n = 4). * p<0.05 versus control cells. # p<0.05 versus HMB treated cells.</p

HMB induces neurite outgrowth in Neuro2a cells.

<p><b>(A)</b> Neuro2A cells were incubated with 25 μM HMB during 72 h and cell viability was measured at different periods of time. <b>(B)</b> Neurite outgrowth was analyzed after 48 h incubation with HMB. Cell morphology was observed under a light microscope (200x) and the neurite bearing cells were counted. Results are plotted as percentage of neurite bearing cells. <b>(C)</b> Acetylcholinesterase activity was measured after 48 h incubation with HMB. <b>(D)</b> Acetylcholinesterase amount was measured by western blot using specific antibodies against AChE. after 48 h incubation with HMB. Results were normalized using actin as loading control. Results are expressed as means ± SEM (n = 4). * p<0.05 versus control cells.</p

Effects of HMB supplementation on morphometric analysis and hind limb strength in DEX-treated rats.

<p><b>(A)</b> Muscle fiber cross-sectional area (μm²). Measurements were made 21 days post treatment. <b>(B)</b> Hind limb strength in animals submitted to saline, DEX or HMB/DEX. Values are expressed as mean ± SEM (n = 7). ^ap<0.05 compared with Control group. ^bp<0.05 compared with DEX group.</p

Effects of HMB on Ub-C promoter activity and expression of Atrogin-1 and MuRF1 induced by DEX.

<p><b>(A)</b> Cells were transiently transfected with an UbC-luciferase reported plasmid and differentiated into myotubes before treatments to evaluate UbC gene transcription (n = 6). Western blots for Atrogin-1 <b>(B)</b> and MuRF1 <b>(D)</b>. Densitometric quantification of protein abundance using actin as a reference control is shown. Signal densities from untreated cells were assigned a value of 100% (n = 4). mRNA levels for Atrogin-1 <b>(C)</b> and MuRF1 <b>(E)</b> were measured in samples from all the experimental groups (n = 8). Results are expressed as means ± SEM. ^ap ‹ 0.05 compared with untreated cells and ^bp ‹ 0.05 compared with DEX-treated cells.</p

Effects of HMB supplementation on body weight, lean body mass and muscle weight in DEX-treated rats.

<p><b>(A)</b> Body weight and <b>(B)</b> lean body mass analysis of each group (Control, DEX and HMB/DEX). Day 0 was considered as the day of initiation of dexamethasone treatment. Wet weight (grams) of soleus <b>(C)</b> and gastrocnemius <b>(D)</b> for each group at the end of the experiment. Values are expressed as mean ± SEM (n = 7). ^ap<0.05 compared with Control group. ^bp<0.05 compared with DEX group.</p

Effects of HMB supplementation on the autophagosome formation induced by DEX.

<p>Autophagosomes were quantified by counting GFP-LC3 punctae/cell of at least 4 fields per treatment. Scale bar in the microphotographs corresponds to 25 μm. Results represent means ± SEM (n = 4).^a p < 0.05 compared with untreated control cells; ^b p < 0.05 compared with DEX-treated cells.</p

Effects of HMB supplementation on the regulation of LC3, p62, Bnip3 and S6K1 expression induced by DEX.

<p><b>(A)</b> Immunoblotting for LC3 in absence or presence of Bafilomycin A1. Two bands are shown, corresponding to LC3-I and the lipidated form LC3-II. <b>(B)</b> Levels of p62 were evaluated by immunoblot analysis in the absence or presence of 100 nM Bafilomycin A1. <b>(C)</b> mRNA levels for Bnip3 were measured by quantitative real-time PCR analysis in samples from all the experimental groups (n = 8). <b>(D)</b> Phosphorylation level of S6K1. Signal densities from untreated cells were assigned a value of 100%. Results are expressed as means ± SEM (n = 4). ^a p ‹ 0.05 compared with untreated cells and ^b p ‹ 0.05 compared with DEX-treated cells.</p

Effects of HMB on the phosphorylation of FoxO3a, Akt and ERK1/2 and on FoxO transcriptional activity in presence of DEX.

<p>Cells were lysed and total protein was immunoblotted with specific phospho- and total-antibodies against FoxO3 (<b>A),</b> Akt/PKB (<b>B</b>) or ERK1/2 <b>(C)</b>. Signal densities from untreated cells were assigned a value of 100%. Results are expressed as means ± SEM (n = 4).^a p ‹ 0.05 compared with untreated cells and ^b p ‹ 0.05 compared with DEX- treated cells. <b>(D)</b> Cells were transiently transfected with a FoxO luciferase reported plasmid and differentiated into myotubes before treatments to evaluate FoxO-dependent transcription. Cells were pre-incubated for 30 min with 10 μM PD98059 and 20 μM LY294002, then incubated with 25 μM HMB for 48 h and finally incubated with 5 μM DEX in the presence or absence of effectors. Inhibitors were maintained during the experiment. Results are expressed as means ± SEM (n = 6). ^ap ‹ 0.05 compared with control cells in the presence of each inhibitor, ^bp ‹ 0.05 compared with DEX- treated cells in the presence of each inhibitor and ^cp ‹ 0.05 compared with control cells in the absence of inhibitors.</p