Proliferation and migration in plasma treated fibroblast-like cells.

<p>HSCs were labelled with 25 µM CFSE and incubated with NAC or 5 µg/ml cytochalasin B. Cells were then treated with plasma and incubated for 72 hrs. Panel <b>A</b> outlines representative FACS analysis of cellular proliferation assessed by evaluating the partition of CFSE. Similar results were obtained in nine independent experiments, each performed in triplicate. Panel <b>B</b> reports the percentage of proliferating fluorescent cells. Panel <b>C</b> reports the fluorescence relative to 6-CFDA in cells migrating to the bottom side of trans-well inserts (a.u. means arbitrary unit). Data are reported as mean±SE of results collected in nine independent experiments, each performed in triplicate. ° denotes P<0.02 <i>vs</i> non treated cells. * denotes P<0.05 <i>vs</i> not treated cells. Plasma treated ISEMFs were seeded onto glass coverslips. The cell monolayer was wounded using a plastic tip and 72 hrs later the cells were stained with haematoxylin and eosin. Images were observed and captured using a light transmission microscope connected to a camera (DMLB Leica, Panel <b>D</b>). Similar results were obtained in three independent experiments.</p

Sources of ROS in plasma-treated fibroblast-like primary cells.

<p>HSCs and ISEMFs were loaded with JC-1 and exposed to plasma or to 20 µM H₂O₂. Cells were cultured for 1, 6, 24 and 48 hrs under optimal conditions and mitochondrial dysfunction was evaluated by FACS analysis in 10,000 events (panel <b>A</b>). H₂DCFDA-loaded cells were treated for 15 min with 5 mM <i>N</i>-acetyl L-cysteine (NAC), 20 µM reduced glutathione (GSH), 50 µM α-tocopherol, 1000 U/ml catalase and exposed for 2 min to plasma. After 5 min, the percentage of fluorescence relative to intracellular ROS was evaluated by FACS analysis in 10,000 events (panel <b>B</b>). Cells were treated with plasma for 2 min and incubated under standard conditions for 5 min or 6 hrs. Thiobarbituric acid (TBA)-reactants were evaluated in cell lysates and normalized to protein contents (panel <b>C</b>). Data are reported as mean±SE of results collected in nine independent experiments, each performed in duplicate or triplicate. ° denotes P<0.02 <i>vs</i> non treated cells. ^§ denotes P<0.05 <i>vs</i> non treated cells (CTR). * denotes P<0.05 <i>vs</i> plasma-treated cells.</p

Intracellular signalling of plasma-induced ROS.

<p>Representative Western blot analysis of PPAR-γ in protein extracts from HSCs and ISEMFs treated with plasma and incubated for 4 hrs. As indicated, cells were pre-treated with NAC. Similar results were obtained in three independent experiments. β-actin was used as a loading control (panel <b>A</b>). Cells were treated for 30 min with 50 µM PPAR-γ receptor antagonist T0070907, exposed to plasma, and then incubated under standard conditions for 24 hrs. IL-6 (panel <b>B</b>) and IL-1β (panel <b>C</b>) were assessed in the conditioned medium by ELISA. Data are reported as mean±SE of results collected in nine independent experiments, each performed in triplicate. * denotes P<0.05 <i>vs</i> non treated cells.</p

Schematic representation of helium generated plasma source.

<p>The plasma source used in the present study consisted of two coaxial tubes, the outer one made out of copper and the inner one made out of insulating material. Two parallel grids 1 mm away from one another closed the tubes. The electric field was formed in the space between the two grids by applying a RF voltage difference supplied by a radiofrequency (RF) generator coupled to the source by a matching network.</p

Plasma-induced intracellular ROS levels.

<p>Primary cultured human hepatic stellate cells (HSCs) and intestinal subepithelial myofibroblasts (ISEMFs) were loaded with H₂DCFDA and exposed for 1, 2, 5, or 10 min to helium-generated plasma. The cells were cultured for 5 min under optimal conditions and then evaluated for intracellular ROS generation by FACS analysis. ROS formation was expressed as the percentage of fluorescence relative to intracellular ROS (ROS fluorescence %, panel <b>A</b>). Intracellular ROS generation was evaluated in fibroblast-like primary cells exposed for 2 min at different distances from the plasma source tip (panel <b>B</b>). The gap between the plasma source tip and the sample was fixed at 1.5 mm, and intracellular ROS generation was evaluated at different times of incubation after 2 minutes of plasma treatment or following exposure to 20 µM H₂O₂ (panel <b>C</b>). During FACS analysis 10,000 events were collected. Data are reported as mean±SE of the percentage of fluorescence registered in nine independent experiments, each performed in duplicate or in triplicate. * denotes P<0.05 <i>vs</i> respective not treated cells (CTR).</p

Evaluation of cell death.

<p>Plasma-treated and H₂O₂ exposed cells were cultured for 1, 6, 12, 24, and 48 hrs. Cells were then stained with Annexin-V (A) and propidium iodide (PI) and evaluated by FACS analysis to assess cell death in 10,000 events. Data are reported as mean±SE of results collected in nine independent experiments, each performed in duplicate or triplicate.</p><p>* denotes P<0.05 <i>vs</i> not treated cells.</p

qPCR analysis of selected genes at different time points.

<p>Two human corneas were used to evaluate the expression of specific genes by qPCR at selected time points from the exposure to APCP. To minimize the variability of response, both corneas, one used as negative control and the other exposed to 2 min APCP, were divided into three pieces, then collected at 3, 6 and 24 h post-treatment. Expression levels of the target genes, detected in the treated corneal sample relative to the untreated sample were normalized to GAPDH levels.</p

Gene Ontologies most represented in the corneal genes up-regulated by APCP.

<p>Gene Ontologies most represented in the corneal genes up-regulated by APCP in the absence (a) or presence (b) of NAC. Size and gray scale color of the circles reflect the importance of cell pathways, represented as functionally connected nodes.</p

Expression levels of selected genes as measured by RNAseq and qPCR.

<p>The expression values generated by RNA-seq (red) or qPCR (blu) for eleven selected genes using the same corneal samples (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133173#pone.0133173.t001" target="_blank">Table 1</a>) are compared. Values detected for each gene were normalized to GAPDH expression and reported as a ratio between APCP-exposed samples and unexposed controls.</p

Differentially expressed corneal genes (DEGs) at 6 h after exposure to APCP.

<p>(a) over- and under-expressed DEGs are shown as common (overlapping area) or exclusive to the APCP (left) or APCP+NAC (right) treatments; (b) number of total (Baggerly’s test FDR p-value <0.01) and DEG, over- and under-expressed genes detected in HC1-HC6 samples, paired per condition.</p