Figure 1. Thrombin increases synthesis of Cyr61 by human corneal stromal fibroblasts and myofibroblasts. A-C: Human corneal fibroblasts (Fibros) and myofibroblasts (Myos) were incubated with a serum-free medium alone (-) or a serum-free medium containing 1.0 U/ml thrombin (+) for 60 min. Alterations in the Cyr61 mRNA levels were detected using agarose gels (A) to visualize the reverse transcriptase-PCR (RT–PCR) products of primers in exons 1 and 5 (Table 1) and were quantified by real-time PCR using primers in exons 2 and 3 (Table 1). GAPDH was used for the normalization. Controls were negative. For both types of experiments, n = 4 using cells from individual donors. A representative gel is given in A. The real-time PCR data (B, C) were analyzed with a one-tailed Student t test. Fibroblasts: t = -2.271, df = 14, *p = 0.020, n = 8; myofibroblasts: t = -2.205, df = 10, **p = 0.026, n = 6. Error bars represent standard error of means (SEM). D: Location of the epitope for the central linker region-specific Cyr61 polyclonal antibody used in E-H. E-H: Fibroblasts (E, G) and myofibroblasts (F, H) were incubated in a serum-free medium from 1-7 h with (+) or without (-) thrombin (1.0 U/ml) for E and F. For G and H, the cells were incubated in a serum-free medium (Control) or thrombin (0.01 to 10 U/ml) for 3 h. Cell lysates were collected, processed, and evaluated for Cyr61 via western blot analysis. GAPDH was used as the loading control in E and F and the modified Bradford Coomassie assay was used for G and H. These methods gave equivalent results. Representative blots are shown with corresponding mean band densities from independent experiments (E, F: n = 5 and G, H: n = 3) of different cell donors. E, F: The two-way ANOVA for Time: F = 4.872, df = 3, 32, p = 0.007 for fibroblasts, and F = 6.042, df = 3, 32, p = 0.002 for fibroblasts; for Treatment: F = 102.569, df = 1, 32, p<0.001 for fibroblasts and F = 79.511, df = 1, 32, p<0.001, n = 5 for myofibroblasts, and the Time and Treatment Interaction factor for fibroblasts: F = 5.051, df = 3, 32, p = 0.006, n = 5 and for myofibroblasts: F = 2.658, df = 3, 32, p = 0.065, n = 5. Significant Tukey multiple pairwise comparisons are *p<0.001, **p<0.005, ***p<0.010, ****p = 0.011. Error bars displayed are SEM. G, H: One-way ANOVA for fibroblasts: F = 3.605, df = 4, 10, p = 0.046, n = 3 and for myofibroblasts: F = 17.669, df = 7, 16, p<0.001, n = 3. Significant Dunnett’s multiple pairwise comparisons are *p<0.001, **p = 0.017, ***p = 0.026. Error bars displayed are SEM.