Figure 3. IL-1β regulates endothelial to mesenchymal transition-related gene expression through FGF2 in the mouse corneal endothelium ex vivo. The mouse corneal endothelium ex vivo was transfected with either fibroblast growth factor 2 (FGF2) siRNA or non-targeting control siRNA (NT) and then maintained in organ culture with interleukin-1 beta (IL-1β) or vehicle control (Veh C). Total RNA was isolated at 14 days post-transfection. A: Induction of FGF2 with IL-1β stimulation was knocked down by FGF2 siRNA at up to 14 days post transfection. RT–PCR showed inhibition of IL-1β-dependent expression of Snai1 and Zeb1 by FGF2 siRNA but not with non-targeting (NT) control siRNA. FGF2 one-way ANOVA, F(4,10)=43.4, p=0.002, n=3 per sample. Tukey’s post-hoc test, honestly significant difference (HSD)[0.05]=1.6 and HSD[0.01]=2.1. Snai1 one-way ANOVA, F(4,10)=208.8, p<0.0001, n=3 per sample. Tukey’s post-hoc test, HSD[0.05]=1.1 and HSD[0.01]=1.5. Zeb1 one-way ANOVA, F(4,10)=182.8, p<0.0001, n=3 per sample. Tukey’s post-hoc test, HSD[0.05]=1.6 and HSD[0.01]=2.1. B: IL-1β-dependent expression of Col1a1, Col1a2, fibronectin (Fn1), and vimentin (Vim) was also inhibited by FGF2 siRNA but not non-targeting (NT) control siRNA in the mouse corneal endothelium ex vivo. Suppression of E-cadherin (Cdh1) with the IL-1β treatment was also reversed with FGF2 siRNA knockdown. Col1a1 one-way ANOVA, F(4,10)=68.0, p=0.001, n=3 per sample. Tukey’s post-hoc test, HSD[0.05]=2.3 and HSD[0.01]=3.1. Col1a2 one-way ANOVA, F(4,10)=759.0, p<0.0001, n=3 per sample. Tukey’s post-hoc test, HSD[0.05]=1.0 and HSD[0.01]=1.3. Fn1 one-way ANOVA, F(4,10)=186.7, p<0.0001, n=3 per sample. Tukey’s post-hoc test, HSD[0.05]=0.1 and HSD[0.01]=1.3. Vim one-way ANOVA, F(4,10)=53.9, p=0.001, n=3 per sample. Tukey’s post-hoc test, HSD[0.05]=2.6 and HSD[0.01]=3.4. Cdh1 one-way ANOVA, F(4,10)=122.0, p=0.0002, n=3 per sample. Tukey’s post-hoc test, HSD[0.05]=0.14 and HSD[0.01]=0.19. C: FGF2 siRNA also knocked down IL-1β-dependent expression of Cdk2 and Ccne1. Cdk2 one-way ANOVA, F(4,10)=152.9, p=0.001, n=3 per sample. Tukey’s post-hoc test, HSD[0.05]=1.0 and HSD[0.01]=1.3. Ccne1 one-way ANOVA, F(4,10)=104.8, p=0.0003, n=3 per sample. Tukey’s post-hoc test, HSD[0.05]=1.4 and HSD[0.01]=1.8. D) Expression of Col8a2 and β-actin (Actb) was not affected by siRNA. The data are representative of the results in three experiments. NT, non-targeting control; Veh C, vehicle control. * p<0.05.