Treatment with the antioxidant <i>N</i>-Acetyl-L-Cysteine (NAC) or with the p53 inhibitor, Pifithrin-α (PFT), prevents cell death in hMp84-overexpressing A375 cells.

<p>At 24 h after transfection, in EV- and p84-cells, incubated with or without NAC (5 mM) or PFT (1 μM), cell death was evaluated as percentage of floating cells on total cells (<b>A</b>), as LDH released in the culture medium (<b>B</b>), and as caspase-3/-7 enzymatic activity (<b>C</b>). Results are presented as mean ± S.E. of three (A and B) or five (C) independent experiments. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001; n.s.: non-significant.</p

Gene expression analysis of oxidative stress-related genes in A375 melanoma cells over-expressing hMp84.

<p>Gene expression was analyzed in triplicate samples by using RT² Profiler PCR Array—Human Oxidative Stress. For <i>NCF2</i>, <i>GSTZ1</i> and <i>CCL5</i>, three additional independent experiments in triplicates were performed by RT-PCR. Gene expression values in p84-cells are expressed as fold-change (mean ± S.E.) over EV-cells after 24 h of transfection.</p><p>*SOD2 has been included both in genes involved in Reactive Oxygen Species (ROS) production/metabolism and in oxidative stress-responsive genes, encoding antioxidants.</p><p>Gene expression analysis of oxidative stress-related genes in A375 melanoma cells over-expressing hMp84.</p

hMp84 induces DNA damage and oxidative alterations of phospholipids in A375 melanoma cells, by stimulating the formation of Reactive Oxygen Species (ROS).

<p>In EV-cells and in p84-cells at 24 h after transfection, DNA damage (by means of Comet assay) (<b>A</b>) and total (esterified <i>plus</i> non-esterified) F₂-isoprostanes were measured (<b>B</b>). In intact EV-cells and p84-cells, the formation of ROS was measured in the intracellular milieu (by DCF-based assay) (<b>C</b>), or in the extracellular milieu (by A6550-based assay) with or without the NADPH-oxidase inhibitor, DPI (10 μM), added in the assay mixture (<b>D</b>). Results are presented as mean ± S.E. of two independent experiments performed in duplicate (DNA comets), four independent experiments (F₂-isoprostanes), or six independent experiments (ROS formation). Results in panel D are presented as mean ratio (over EV-cells) ± 95% confidence interval of three independent experiments. *<i>P</i><0.05, **<i>P</i><0.01, and ***<i>P</i><0.001, compared to control EV-cells. n.s.: non-significant, compared to p84-cells without DPI.</p

Treatment with the antioxidant <i>N</i>-Acetyl-L-Cysteine (NAC) or with the p53 inhibitor, Pifithrin-α (PFT), prevents ROS formation in hMp84-overexpressing A375 cells.

<p>At 12 h (<b>A</b>) and 24 h (<b>B</b>) after transfection, ROS formation was evaluated by DCF-based assay in EV-cells and p84 cells, incubated with or without NAC (5 mM) or PFT (1 μM). Results are presented as mean ± S.E. of six (A) or three (B) independent experiments. *<i>P</i><0.05; **<i>P</i><0.01; n.s.: non-significant.</p

Over-expression of hMp84 mutated in the active site (hMp84^C42S) does not affect cell fate in both A375 and HT-144 melanoma cells.

<p>All parameters were evaluated at 24 h after transfection. hMp84^C42S protein was evaluated by Western blot in adhering and floating cells of A375- (<b>A</b>) and HT-144-transfected cells (<b>B</b>); typical Western blots out of three are shown. Cell proliferation (total cell number) was evaluated in parental, non-transfected cells (W), in EV- and in hMp84^C42S-cells of A375 (<b>C</b>) and HT-144 cells (<b>D</b>). Cell detachment was evaluated in EV- and in hMp84^C42S-cells of A375 (<b>E</b>) and HT-144 cells (<b>F</b>). Results are presented as mean ± S.E. of at least three independent experiments. NS = non-significant and *<i>P</i> = 0.046 compared to EV-cells.</p

Transient over-expression of Calpain-3 (variant hMp84) impairs cell proliferation and leads to cell detachment in A375 melanoma cells.

<p>At 24, 48 and 72 h after transfection, hMp84 protein (p84) was evaluated by Western blot in empty vector- (EV) and in hMp84-transfected cells (p84), both in still adhering, viable cells (<b>A</b>) and in floating cells (<b>B</b>), separately harvested. A typical Western blot out of three is shown. At the same times after transfection, cell proliferation (total cell number) (<b>C</b>) and cell detachment (% of floating cells on total cells) (<b>D</b>) were evaluated in parental, non-transfected cells (W), in EV-cells and in p84-cells. Results are presented as mean ± S.E. of at least four independent experiments. *<i>P</i><0.05; **<i>P</i><0.01.</p

hMp84 stimulates the formation of Reactive Oxygen Species (ROS) and induces oxidative alterations of phospholipids in HT-144 melanoma cells.

<p>At 24 h after transfection, the formation of intracellular ROS (by DCF-based assay) (<b>A</b>) and total (esterified <i>plus</i> non-esterified) F₂-isoprostanes (<b>B</b>) were measured in adhering EV- and p84-cells. Results are presented as mean ± S.E. of three to five independent experiments. *<i>P</i><0.05.</p

Erythrocyte membrane beta-actin band decrease intensities in RTT patients.

<p>Result indicate band intensities as expressed as relative %V normalized values by SDS-PAGE analysis. Data are mean±SD.</p>a<p>Band numbers refer to those of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093181#pone-0093181-g001" target="_blank">Figure 1</a>. Bold characters indicate the beta-actin band intensity.</p><p>** = <i>P</i><0.01.</p

Potential binding sites for 4-HNE in the beta-actin amino acid sequence.

<p>The potential binding sites for 4-HNE are highlighted. Red color for 6 cysteine (C), yellow color for 8 hystidine (H) and green color for 19 lysine (K) residues. Blue double lines indicate sub-domain 1; orange lines indicate sub-domain 2; black lines indicate sub-domain 3 and purple lines indicate sub-domain 4 (primary sequence extracted from the ExPASy: SIB Bioinformatics Resource Portal, <a href="http://www.expasy.org/" target="_blank">http://www.expasy.org/</a>).</p

Western blot of beta-actin.

<p>Comparative analysis of RBC ghosts from healthy controls and RTT patients using four antibodies, which recognize different beta-actin amino acidic sequences (A: full length, B: N-terminal, C: C-terminal and D: C-terminal, amino acidic sequence not specified), confirm a beta-actin decrease in RTT patients.</p