Involvement of AIF in CSE-induced Apoptosis.

<p>PBECs were left untreated (A), or were treated for 24h with 20% CSE alone (B), or with a combination of CSE and GSH (1mM) (C) or AA (250nM) (D). Cells were then washed, fixed and stained with a primary antibody directed towards AIF, followed by a secondary FITC-conjugated antibody. The figure shows that in basal conditions, PBEC expressed AIF in their cytoplasm especially inside their mitochondria (A, insert); CSE treatment caused translocation of AIF from the mitochondria to inside the nuclei (B, green arrows and insert); GSH pre-treatment caused a significant decrease in the number of nuclear-AIF positive cells whereas AA was not so effective. Bar = 30μm. Data are representative of experiments performed with PBECs from 2 donors.</p

Effect of Caspase Inhibitors on CSE-Induced Apoptosis.

<p>PBEC obtained from 4 non-asthmatic and 4 asthmatic donors were treated for 24h with 20%CSE alone or in combination with Ac-DEVD-CHO (C3i) (a) or Z-LEHD-FMK (C9i) (b). Changes in EA were evaluated with AxV staining. The results are displayed as a box plot showing median and 5–95% confidence intervals of fold changes from CSE only in both groups.</p

Ability of GSH and AA to protect against H₂O₂-induced cell death.

<p>PBEC obtained from non-asthmatic or asthmatic donors were treated for 24h with 400μM H₂O₂ alone or in the presence of GSH (1mM) or AA (250nM). Changes in cell viability (a) and EA (b) were then evaluated with AxV staining. The results are displayed as a box plot showing median, interquartile range and 5–95% confidence intervals of fold changes from H₂O₂ alone for PBECs from 10 non-asthmatic (○) and 10 asthmatic (Δ) donors. * = p<0.02 according to Wilcoxon Signed Rank tests comparing antioxidants with H₂O₂ only. There was no statistical difference comparing antioxidants treatment between PBECs from non-asthmatic or asthmatic donors according to a Mann Whitney U test.</p

Ability of GSH, but not AA, to protect against CSE-induced cell death.

<p>PBEC obtained from non-asthmatic or asthmatic donors were treated for 24h with 20% CSE alone or in the presence of GSH (1mM) or AA (250nM). Changes in cell viability (a) and EA (b) were then evaluated with AxV staining. The results are displayed as a box plot showing median, interquartile range and 5–95% confidence intervals of fold changes from CSE alone using PBECs from 10 non-asthmatic (○) and 10 asthmatic (Δ) donors. <b>*</b> = p<0.05 according to Wilcoxon Signed Rank tests comparing GSH+CSE with CSE only. <b>∞</b> = p<0.05 according to a Mann Whitney U test comparing CSE+GSH treatment between PBECs from non-asthmatic and asthmatic donors.</p

Effect of CSE on PBEC Viability and Apoptosis.

<p>PBEC obtained from non-asthmatic and asthmatic donors were treated for 24h with 20% CSE. Cell viability (a) and early apoptosis (b) were then evaluated with AnnexinV staining. The results are displayed as a box plot showing median, interquartile range and 5–95% confidence intervals using PBECs from 10 non-asthmatic (○) and 10 asthmatic (Δ) volunteers. * represents significance (p<0.05) according to Wilcoxon Signed Rank tests comparing treatment with untreated control. ∞ represents significance (p = 0.003) according to a Mann Whitney U test comparing CSE treatment between PBECs from non-asthmatic and asthmatic donors.</p

Characteristics of subjects with asthma.

<p>¹Inhaled corticosteroid (ICS) dose is given as μg of beclomethasone (BDP) used per day, expressed as the mean (sd).</p><p>²Values for FEV₁ as a percentage of the predicted FEV₁ are given as a mean and standard deviation (sd).</p><p>Characteristics of subjects with asthma.</p

Effect of CLA supplementation on body weight, skeletal muscle, strength, and endurance performance <i>in</i><i>vivo</i>.

<p>A. Changes in body weight over time. B. Weight of the hindlimb anterior muscle groups (tibialis anterior, extensor hallucis longus, and extensor digitorum longus) normalised for body weight. C. Strength of the forelimb. D. Strength of the forelimb normalised to the body weight. E. Distance ran during the endurance test. SED, sedentary; TR, trained; PLA, placebo; CLA, conjugated linoleic acid. # Significant difference compared to time point “0” (P<0.05). ◊ Significant difference compared to the CLA-SED group (P<0.05). $ Significant difference compared to the PLA-SED group (P<0.05).</p

Effect of CLA supplementation on steroidogenic genes and proteins <i>in</i><i>vitro</i>.

<p>A. Real-time PCR analysis of genes encoding steroidogenic enzymes from <i>in </i><i>vitro</i> R2C cell extract. The graph shows the normalisation with the reference genes, according to the Livak Method (2-∆∆CT). B. Representative western blots showing CYP17A1 (55 kDa) and ß-actin (42 kDa) expression following electrophoretic separation of protein extracts obtained from R2C cells treated with different concentrations of CLA (w/o, 0.5, 1.5 and 7.5 µM CLA). C. Relative amounts of CYP17A1. † Significant difference compared to w/o CLA (P<0.01). </p

Effect of CLA supplementation on testosterone biosynthesis, perilipin A/C, and hormone sensitive lipase (HSL) levels in R2C cells.

<p>A. Total testosterone biosynthesis following treatment with different CLA concentrations (w/o, 0.5, 1.5 and 7.5 µM CLA). B. Representative western blots showing HSL (84 kDa), perilipin A/C (58 and 42 kDa, respectively), and ß-actin (42 kDa) expression following electrophoretic separation of protein extracts obtained from R2C cells treated with different concentrations of CLA. C. Relative amounts of HSL, ß-actin, perilipin A and C. D. Representative immunofluorescent images of perilipin A/C expression in R2C cells treated with different concentrations of CLA. E. Representative immunofluorescent images of HSL expression in R2C cells treated with different concentrations of CLA. † Significant difference compared to w/o and 0.5 µM CLA (P<0.01).</p

GA affected HSPs protein levels in OS 143B cells.

<p>OS 143B cells were treated with 4 µM GA for 6 h; the levels of Hsp60, Hsp70, total cellular Hsp90, Hsp90AA1, Hsp90AB1proteins were determined by Western blotting. <b>A</b>. GA decreased the level of Hsp60 and induced post-translational modification of Hsp60 partially derived from the hyperacetylated isoform. <b>B–E</b>. GA upregulated Hsp70 (B), total cellular Hsp90 (C), Hsp90AA1 (D), Hsp90AB1 (E) protein levels. Each experiment was performed at least three times. The representative data are shown. Densitometric analysis of HSP/beta-actin was performed using Quantity one 4.5.2 software.</p