Fig 2: Regulation of FGF2-induced mesenchymal-related gene expression by SP3 is limited in human corneal endothelium ex vivo. (A) SP3 siRNA knockdown led to a decrease in FGF2-dependent SP3 expression but only a slight inhibition of FN1, VIM, COL1A1, and COL1A2 expression in human ex vivo corneal endothelium. SP3 F(4,10) = 48.2, P < 0.0001, n = 3 per sample. Tukey's post hoc test, HSD[0.05] = 1.8 and HSD[0.01] = 2.4. FN1 F(4,10) = 372.7, P < 0.0001, n = 3 per sample. Tukey's post hoc test, HSD[0.05] = 0.4 and HSD[0.01] = 0.5. VIM F(4,10) = 125.8, P < 0.0001, n = 3 per sample. Tukey's post hoc test, HSD[0.05] = 0.8 and HSD[0.01] = 1.0. COL1A1 F(4,10) = 130.3, P < 0.0001, n = 3 per sample. Tukey's post hoc test, HSD[0.05] = 0.5 and HSD[0.01] = 0.7. COL1A2 F(4,10) = 128.5, P < 0.0001, n = 3 per sample. Tukey's post-hoc test, HSD[0.05] = 0.6 and HSD[0.01] = 0.8. (B) SP3 siRNA knockdown led to a decrease in FGF2-dependent SP3 protein level but only slight decreases in FN1, VIM, COL1 proteins. Transfection with nontargeting control siRNA did not alter gene expression in all experiments. COL8A2 and β-actin were used as a corneal endothelial marker and loading control, respectively. Veh C, vehicle control; NT, nontargeting control; FN1, fibronectin; VIM, vimentin; ACTB, β-actin.

Fig 3: 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 4: Binding between SP1 and SP3 proteins in human CECs treated with FGF2. Immunoprecipitation (IP) with anti-SP1 or anti-SP3 antibodies was performed on extracts prepared from human CECs treated with FGF2. Resulting complexes were separated on polyacrylamide gels and immunoblotted with the other anti-SP3 or anti-SP1 antibodies (IB). Binding between SP1 and SP3 in FGF2 stimulated human CECs was demonstrated by coimmunoprecipitation. SP1 and SP3 were not detected in cells treated with vehicle control. β-actin was used as a loading control. IP, immunoprecipitation; IB, immunoblotting; Veh C, vehicle control; F2, FGF2.

Fig 5: FGF2 induces SP3 expression through a ZEB1-independent pathway in human corneal endothelium ex vivo. (A) After FGF2 stimulation for indicated number of days times, total RNA and protein were purified from human corneal endothelium ex vivo for RT-PCR (left panel) and immunoblotting (right panel). FGF2 treatment led to an increase in SP3 expression in a time-dependent manner. D, days. (B) ZEB1 siRNA knockdown did not affect the FGF2-dependent expression of SP3 at either the transcriptional (left panel) or translational (right panel) level. SP3 F(4,10) = 24.6, P < 0.0001, n = 3 per sample. Tukey's post hoc test, HSD[0.05] = 2.3 and HSD[0.01] = 3.0. ZEB1 F(4,10) = 87.6, P < 0.0001, n = 3 per sample. Tukey's post hoc test, HSD[0.05] = 1.0 and HSD[0.01] = 1.3. COL8A2 was used as a corneal endothelial marker, and β-actin used as a loading control. Veh C, vehicle control; NT, non-targeting control; ACTB, β-actin.