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. 2018 Mar 13;8:4438. doi: 10.1038/s41598-018-22821-9

Figure 4.

Figure 4

Evaluation of the combined effects of TGF-β1 and EGF on the morphology and the phenotype of corneal endothelial cells in the proliferation and the maturation phases. (a) Proliferation phase: Phase contrast images (left column), immunofluorescence detection of ZO-1 (second column), N-Cadherin (middle column) and α-SMA (right column) of CECs cultured in a proliferation medium alone (P) or supplemented with TGF-β1 or a combination of TGF-β1 and the EGFR inhibitor AG-1478. Note that the proliferation medium contains 5 ng/ml of EGF. (b) Maturation phase: CECs were cultured in the proliferation medium until confluency then cultured for 7 days in the maturation medium alone (M) or supplemented with TGF-β1 or a combination of TGF-β1 and EGF, or a combination of TGF-β1, EGF and AG-1478. Note that the basal maturation medium does not contain EGF. (c) Left: Representative Western blot of n-cadherin from CECs at confluency (P-conditions) or at 7-days post-confluency (M-conditions). α-tubulin served as a loading control. Cropped images were displayed and full-length blots are shown in the Supplementary Figure S6a. Right: Relative quantification of the n-cadherin Western blots to the α-tubulin loading control, as described in the Materials and Methods. Mean of all 4 experiments (1 sample/condition/cell population) were calculated with standard deviation. Cell populations were separated in the same subgroups of high and low basal expression of α-SMA and coll.I (described in d-e). Two-way ANOVA followed by Tukey’s multiple comparison tests were performed. (d–e) Analysis of immunofluorescence micrographs for endothelial-to-mesenchymal transition markers. The relative expression of α-SMA (d) to f-actin and type I collagen (e) to the number of nuclei was calculated in immunofluorescence micrographs (9 per condition). Results are presented as mean ± S.D. Cell populations in d and e were separated in two subgroups according to their basal protein expression in the proliferation control condition. Low basal expression of α-SMA (d) and coll.I (e) (black bars); high basal expression of α-SMA (d) and coll.I (e) (grey bars). Two-way ANOVA followed by Tukey’s multiple comparison tests were performed. (f) Left: Representative Western blot of α-SMA (e) from CECs at confluency (P conditions) or at 7-days post-confluency (M-conditions). α-tubulin served as a loading control. Cropped images were displayed and full-length blots are shown in the Supplementary Figure S6b. Right: Relative quantification of the α-SMA Western blots to the α-tubulin loading control, as described in the Materials and Methods. Mean of all 4 experiments (1 sample/condition/cell population) were calculated with standard deviation. Cell populations were separated in the same subgroups than in d-e. Two-way ANOVA followed by Tukey’s multiple comparison tests were performed. *p < 0,05; **p < 0.005; ***p < 0.0005; ****p < 0.0001. α-SMA: α-smooth muscle actin; α-tub: α-tubuline; Coll.I: type I collagen; EGF: Epidermal growth factor; n-cad: n-cadherin; TGF-β1: Transforming growth factor β1.