Figure 2. FGF2 regulates endothelial to mesenchymal transition–related gene expression in a time-dependent manner in mouse corneal endothelium ex vivo. Fifteen ex vivo corneas from nonobese diabetic (NOD; A, B, C, and D) and C57BL/6 (E, F, and G) mice were cultured for the indicated times (0, 1, 2, 3, or 7 days) with fibroblast growth factor 2 (FGF2). The endothelium–Descemet’s membrane complex was isolated, and total RNA was purified for reverse transcriptase PCR ( RT–PCR). A: Increased expression of Snai1 and Zeb1 was noted in the FGF2-treated mouse corneal endothelium ex vivo as early as 2 days post-treatment. B: The FGF2 treatment led to increased expression of Col1a1, Col1a2, fibronectin (Fn1), and vimentin (Vim), and suppression of E-cadherin (Cdh1) in a time-dependent manner in the mouse corneal endothelium ex vivo. C: Increased expression of Cdk2 and Ccne1 were also noted in FGF2-treated corneas in a time-dependent manner. D: The expression of Col8a2, a corneal endothelial marker, and β-actin (Actb), loading control, was not affected by FGF2. The ex vivo corneal endothelium from the C57BL/6 mouse strain was used to determine whether there were any strain differences in the FGF2 response. After being cultured for 7 days with FGF2, total RNA from mouse corneal endothelium ex vivo was purified, and RT–PCR was performed. E: Marked induction of the endothelial to mesenchymal transition markers Snai1 and Zeb1 was observed in the FGF2-treated but not in the vehicle control (Veh C) mouse corneal endothelium ex vivo. F: FGF2 treatment led to increased expression of Col1a1, Col1a2, fibronectin (Fn1), and vimentin (Vim), and suppression of E-cadherin (Cdh1) in the mouse ex vivo corneal endothelium. G: Increased expression of Cdk2 and Cyclin E1 (Ccne1) was noted in the FGF2-treated but not in the control mouse corneal endothelium ex vivo. Col8a2 and β-actin (Actb) expression was not affected by FGF2. The data shown are representative of the results in three independent experiments. Veh C, vehicle control.