Table 1.
Primers for q-PCR.
Fig 1.
Establishment of responder and non-responder BTM cell strains.
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.
Table 2.
The IOP change in the fellow eye of DEX responder (R) and non-responder (N) TM cell strains.
Fig 2.
Differential induction of Fibronectin (FN) by DEX in responder and non-responder BTM cells.
Confluent BTM cells were treated with 0.1% EtOH or 100nM DEX for 7 days. Conditioned medium was collected for WB. R: responder BTM cells. N: non-responder BTM cells.
Fig 3.
Diagram of differential expression groupings (DEG).
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.
Fig 4.
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 (S3 Table), they are shown in two plots, (A) and (B), respectively. (C) DEG#4; (D) DEG#5.
Fig 5.
Validation of RNA sequencing findings using qPCR.
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 Fig 3. 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.
Table 3.
Three of the most up-regulated and down-regulated genes in DEGs 3–5.
Fig 6.
Pathways associated with DEGs 3, 4, and 5.
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.
Table 4.
Summary of differential microarray gene expression studies in TM cells/tissues.