Fig 2: SP1 and SP3 bind to the promoters of FN1, VIM, and COL1A1 in human CECs treated with FGF2. (A) Illustration of the putative SP1/SP3 binding sites in the promoters of COL1A1, FN1 and VIM. The location of each primer used in the ChIP assay is indicated. (B) The expected PCR product sizes for the primer pairs were as follows: COL1A1, 271 bp; FN1, 253 bp; and VIM, 288 bp. Stimulation with FGF2 resulted in SP1 and SP3 binding to the COL1A1, FN1 and VIM promoters in human CECs. Anti-histone H3 antibody was used as positive control (+) and normal rabbit IgG was used as negative control (-). Veh C, vehicle control; F2, FGF2; FN1, fibronectin; VIM, vimentin.

Fig 3: FGF2 induces SP1 expression through ZEB1 in human corneal endothelium ex vivo. Fourteen days after ZEB1 siRNA transfection of human ex vivo corneas, total RNA and protein were isolated from the endothelium. (A) RT-PCR and (B) immunoblotting showed ZEB1 siRNA knockdown inhibited FGF2-dependent expression SP1. Transfection with nontargeting control siRNA did not alter SP1 expression. Vehicle control did not alter SP1 expression. β-actin was used as a loading control and COL8A2 was used as a corneal endothelial marker. Veh C, vehicle control; NT, non-targeting control.

Fig 4: The proposed regulatory pathway for FGF2 mediated mesenchymal transition in human corneal endothelial cells. Stimulation by FGF2 leads to increased expression of SP1 and SP3. SP1 expression is regulated through ZEB1 whereas SP3 expression is not. SP1 plays a greater role in expression of COL1, FN1 and VIM than SP3.

Fig 5: SP1 and SP3 bind independently to the promoters of FN1, VIM, and COL1A1 in human CECs treated with FGF2. After transfection with SP1 or SP3 siRNA, ChIP assays were performed with anti-SP1 or anti-SP3 antibodies. While siRNA knockdown of either SP1 or SP3 reduced their respective binding to FN1, VIM, and COL1A1 promoters, SP1 knockdown had no effect on SP3 binding and SP3 knockdown had no effect on SP1 binding. Non-targeting control siRNA did not alter their binding activity. Anti-histone H3 antibody was used as positive control (+) and normal rabbit IgG was used as negative control (−). IP, immunoprecipitation; Veh C, vehicle control; NT, non-targeting control; FN1, fibronectin; VIM, vimentin; ACTB, β-actin.