Figure 2
Effect of TGF-β on extracellular matrix (ECM) protein expression and unfolded protein formation. (a) Descemet’s membranes including CECs were obtained from 22 patients with FECD during Descemet’s membrane endothelial keratoplasty (DMEK) and were examined for the expression of ECM; fibronectin, collagen type 1, collagen type 3, and TGBI were evaluated by immunofluorescent staining. Human donor corneas from 19 non-FECD subject were used as control. Scale bar: 50 µm. (b,c) iFECD, which is a corneal endothelial cell model established from patients with FECD, were cultured and stimulated with TGF-β2 for 48 hours. The expression of fibronectin and collagen type 1 was evaluated by immunofluorescence staining. Unfolded protein was assessed by aggresome staining. Nuclei were stained with DAPI. Experiments were performed in triplicate. Scale bar: 50 µm. (d) Immunofluorescent staining of PDI and aggresome staining was performed to evaluate the codistribution of endoplasmic reticulum (ER) and unfolded protein. Nuclei were stained with DAPI. Experiments were performed in triplicate. Scale bar: 50 µm. (e) iFECD and iHCEC were cultured with or without TGF-β2 (10 ng/ml) for 48 hours. Cells were stained with aggresome and fluorescence intensity was evaluated by flow cytometry. Experiments were performed in triplicate. The statistical analysis was performed by Student’s t-test. *P < 0.01. (f) iFECD and iHCEC were cultured with or without TGF-β2 (10 ng/ml), and expression of PERK, phosphorylation of PERK, and expression of CHOP was evaluated by western blotting. Experiments were performed in triplicate. GAPDH was used as internal control.
