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

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

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

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

APCP treatment elicited ROS-dependent adaptive responses in ex-vivo corneas.

<p>Human corneal tissues exposed for 2 minutes to APCP were kept wet at 37°C. At different time periods after exposure, tissues were dissected and snap frozen for RNA and protein extraction. The mRNA transcript levels specific for human glutathione peroxidase 1 (GPX1, panel A) and glycosylase OGG1 (panel B) were evaluated by quantitative real-time PCR. Expression of the target genes was normalized to the endogenous levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data are expressed as mean ± SE of gene copy number in 5 µg of loaded total RNA obtained from two experiments. ^*<i>P</i><0.05 <i>vs</i> untreated cells. Protein expression of GPX and OGG1 was evaluated by Western blot analysis (panel C).</p