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

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

Detection of OGG1 in human corneas treated ex-vivo with APCP.

<p>Corneal tissues exposed for 2 min to APCP were analyzed by immunohistochemistry (a-d) and Western Blot (e-f). Frozen sections (5 μm) of corneas treated in the absence (a) or presence (b) of 10 mM NAC were incubated with polyclonal rabbit anti-OGG1 at 6 h post-treatment. Protein immunostaining (in red) was compared to that of untreated controls (c). Negative controls were prepared by omitting the primary antibody (d). For the Western Blot analysis, proteins were extracted at 6 and 24 h post-treatment: the OGG1 protein signal increased at 6 h, and was reduced in the presence of NAC, and returned to values comparable to that of controls within 24 h. Densitometric values of OGG1 autoradiographic bands were normalized to corresponding β-actin and expressed as percentage ± SE of the mean control value.</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

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

Gene ID, Ensemble ID and related Forward and Reverse primers used in qPCR.

<p>GAPDH was used as housekeeping gene, other genes were selected for RNA-seq validation (underlined) or time-related expression analysis (*)</p><p>Gene ID, Ensemble ID and related Forward and Reverse primers used in qPCR.</p

A selection of corneal genes to this study.

<p>A selection of DEGs for at least one treatment or genes selected for RNA-seq validation (^) or time-related expression analysis (*). Where genes are DEG, fold change value and absolute ranks in the transcriptome was reported. FC, Fold Change.</p><p>A selection of corneal genes to this study.</p

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

Hierarchical clustering performed on the whole normalized dataset.

<p>52,447 genes, Manhattan correlation, complete linkage.</p

Corneal samples used and RNA sequencing data report.

<p>Individual (HC2, HC4, HC6) or pooled (HC1, HC3, HC5) human corneas untreated or treated for 2 min with APCP in the absence or presence of N-acetyl cysteine (NAC) were subjected to RNA sequencing. HC1A-HC1B, HC3A-HC5A, HC3B-HC5B, HC4A-HC6A are cornea pair from the same donor Percentage of obtained Illumina reads on total and mapped reads per sample are shown.</p><p>Corneal samples used and RNA sequencing data report.</p