Figure 2: Inhibition of Zeb1 expression by intracameral 4-OHT injection in the corneal endothelium of Zeb1^flox/flox: UBC-CreERT2 mice. (a) Seven days after intracameral 4-OHT (+) or vehicle (−) injections, the corneal endothelium from wildtype (WT), UBC-CreERT2, Zeb1^flox/flox, and Zeb1^flox/flox: UBC-CreERT2 mice, 6 mice per group, were isolated and maintained in organ culture with FGF2 or vehicle (Con) for 7 days ex vivo. Total RNA was isolated from the corneal endothelium and RT-PCR was performed for Zeb1, Col8a2, ActB, and Ktcn. FGF2 but not vehicle treatment induced Zeb1 expression in all groups as probed by using the forward primer located in exon 6 and the reverse primer located in exon 7. Intracameral injection of 4-OHT in Zeb1^flox/flox: UBC-CreERT2 led to loss of exon 6 in the Zeb1 mRNA in the corneal endothelium. Col8a2 and Actb were used as CEC marker and loading control, respectively. Keratocan (Ktcn) was used to control for possible stromal keratocyte and epithelial cell contamination. (b) Seven days after intracameral 4-OHT (+) or vehicle (−) injections, the corneal endothelium from wildtype (WT), UBC-CreERT2, Zeb1^flox/flox, and Zeb1^flox/flox: UBC-CreERT2 mice, 18 mice per group, were isolated and maintained in organ culture with FGF2 or vehicle (Con) for 7 days ex vivo. Total protein was purified from the corneal endothelium and immunoblotting for Zeb1 and β-actin was performed. FGF2 but not vehicle treatment induced Zeb1 expression in the corneal endothelium of all groups except Zeb1^flox/flox: UBC-CreERT2 mice that received intracameral 4-OHT injection. Zeb1 protein was not detectable in the corneal endothelium of Zeb1^flox/flox: UBC-CreERT2 mice that received intracameral 4-OHT injection indicating targeting of Zeb1 in the corneal endothelium following 4-OHT injection