Fig 2: Verification of collagen transcripts that were upregulated the most in the late phase of conjunctival wound healing by qPCR. (A) Real-time PCR analyses of the 10 most highly expressed collagen genes in mouse conjunctival tissues harvested 2, 7, 14 and 21 days after experimental surgery. Data are calculated as fold-change relative to paired unoperated controls. The mean fold-change of five groups, each consisting of operated tissues pooled from three mice, for each time point is indicated (n=5). *P<0.05 for fold-change compared with all other time points; dP<0.05 for fold-change on day 7 compared with both days 14 and 21; ?P<0.05 for fold-change on day 7 compared with day 21 only. (B) Immunoblot analyses of COL8A1, COL11A1 and COL1A1 in mouse conjunctival tissues harvested on day 2 and day 7 post-experimental surgery. Three paired groups for each time point are shown (n=3). Op, operated tissues pooled from five independent eyes per group; C, paired untreated controls pooled similarly. Fold-change in expression in operated relative to control tissues, both normalized to GAPDH, and the associated P values corrected by Bonferroni adjustment, where significant, are shown below the respective immunoblot.

Fig 3: Induction of top collagen genes by TGFß2 in primary human conjunctival fibroblasts and in individuals with surgical failure. (A) Real-time PCR analyses of COL8A1, COL11A1, COL8A2 and COL1A1 in primary human conjunctival fibroblasts stimulated with TGFß2 for 72 h. Data shown are representative of three independent experiments, and calculated as mean fold-change±s.d. relative to untreated controls. *P<0.05 for fold-change in treated versus control. (B) Immunoblot analyses of COL8A1, COL11A1 and COL1A1 in human fibroblasts treated as indicated for 72 h. Three independent sets of experiments are shown. Fold-change in expression in TGFß2-treated relative to control cells, both normalized to GAPDH, and P values (corrected by Bonferroni adjustment) where significant, are shown in the densitometric analyses below the immunoblot. (C) Real-time PCR analyses of COL8A1 and COL1A1 in human conjunctival tissues from individuals requiring repeat surgeries (n=15) relative to those with no prior GFS (n=20). Data for COL8A1 in tissues of individuals with no prior surgeries was only available from 15 patients as five samples did not produce detectable values. Data shown are calculated as fold-change in expression in tissues of repeat surgery patients relative to that from patients with no prior surgery. Significant fold-changes between the two groups of patients and the associated P values (*) are indicated. Each symbol represents one patient.

Fig 4: Induction of top collagen genes by TGFß2 in primary mouse conjunctival fibroblasts. (A) Real-time PCR analyses of collagen genes in fibroblasts stimulated for 72 h with TGFß2. Data shown are representative of three independent experiments, and show mean fold-change±s.d. relative to untreated controls. *P<0.05 for fold-change in treated versus control. (B) Immunoblot analyses of COL8A1, COL11A1 and COL1A1 in mouse fibroblasts treated as indicated for 72 h. Three independent sets of experiments are shown. Fold-change in expression in TGFß2-treated relative to control cells, both normalized to GAPDH, and P value (corrected by Bonferroni adjustment) where significant, are shown in the densitometric analyses below the immunoblot.