Volcano plot of DEGs 3–5.

<p>The fold of change (log₂) and p value (-log₁₀) of the genes in DEGs 3, 4, and 5 are shown in volcano plots. Since the genes in DEG#3 have two p values, one from responders and the other from non-responders (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169671#pone.0169671.s006" target="_blank">S3 Table</a>), they are shown in two plots, (A) and (B), respectively. (C) DEG#4; (D) DEG#5.</p

A Comparison of Gene Expression Profiles between Glucocorticoid Responder and Non-Responder Bovine Trabecular Meshwork Cells Using RNA Sequencing

<div><p>The most common ocular side effect of glucocorticoid (GC) therapy is GC-induced ocular hypertension (OHT) and GC-induced glaucoma (GIG). GC-induced OHT occurs in about 40% of the general population, while the other 60% are resistant. This study aims to determine the genes and pathways involved in differential GC responsiveness in the trabecular meshwork (TM). Using paired bovine eyes, one eye was perfusion-cultured with 100nM dexamethasone (DEX), while the fellow eye was used to establish a bovine TM (BTM) cell strain. Based on maximum IOP change in the perfused eye, the BTM cell strain was identified as a DEX-responder or non-responder strain. Three responder and three non-responder BTM cell strains were cultured, treated with 0.1% ethanol or 100nM DEX for 7 days. RNA and proteins were extracted for RNA sequencing (RNAseq), qPCR, and Western immunoblotting (WB), respectively. Data were analyzed using the human and bovine genome databases as well as Tophat2 software. Genes were grouped and compared using Student’s t-test. We found that DEX induced fibronectin expression in responder BTM cells but not in non-responder cells using WB. RNAseq showed between 93 and 606 differentially expressed genes in different expression groups between responder and non-responder BTM cells. The data generated by RNAseq were validated using qPCR. Pathway analyses showed 35 pathways associated with differentially expressed genes. These genes and pathways may play important roles in GC-induced OHT and will help us to better understand differential ocular responsiveness to GCs.</p></div

Diagram of differential expression groupings (DEG).

<p>The four groups of raw data (responders and non-responder BTM cells treated with DEX or EtOH) were grouped into 5 DEGs. A) The initial grouping of raw data. DEG #1: DEX vs. EtOH in responders; DEG #2: DEX vs. EtOH in non-responders. B) Further grouping of DEGs 3–5. DEG #3: overlap between DEG groups #1 and 2; DEG #4 = 1–3; DEG #5 = 2–3. C) The number of genes in DEGs#3, 4, and 5.</p

Establishment of responder and non-responder BTM cell strains.

<p>One of the paired bovine eyes was used for perfusion culture, treated with DEX, and IOP was monitored. The fellow eye was directly used to establish a cultured BTM cell strain without prior perfusion culture. Based on the DEX-induced IOP changes in the perfusion cultured eyes (mΔIOP ≥2.82mmHg or <2.82mmHg), the BTM cell strains established from the fellow eyes were defined as responder cell strains or non-responder cell strains, respectively.</p

Primers for q-PCR.

<p>Primers for q-PCR.</p

Pathways associated with DEGs 3, 4, and 5.

<p>C: the number of reference genes in the category; O: the number of genes in the gene set and also in the category; E: the expected number in the category; R: ratio of enrichment; rawP: p value from hypergeometric test; adjP: p value adjusted by the multiple test adjustment. Please be aware that “Up or Down” only refers to whether the genes associated with listed pathways were up or down-regulated. It does not necessarily mean the pathway was activated or inhibited.</p

The IOP change in the fellow eye of DEX responder (R) and non-responder (N) TM cell strains.

<p>The IOP change in the fellow eye of DEX responder (R) and non-responder (N) TM cell strains.</p

Summary of differential microarray gene expression studies in TM cells/tissues.

<p>Summary of differential microarray gene expression studies in TM cells/tissues.</p

Validation of RNA sequencing findings using qPCR.

<p>The same RNA used for RNAseq was used for qPCR. The ΔΔCt method was used for calculation of gene expression changes and actin was used as an internal control. Data analysis/grouping was performed in a similar way as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169671#pone.0169671.g003" target="_blank">Fig 3</a>. Three of the most up-regulated and down-regulated genes of DEG groups 3, 4, and 5 were studied and compared to RNAseq results. Values of Log₂(Fold of change): >0: up-regulation; = 0 non change; <0 down-regulation. n = 3.</p

Three of the most up-regulated and down-regulated genes in DEGs 3–5.

<p>Three of the most up-regulated and down-regulated genes in DEGs 3–5.</p