Figure 4
Effect of chemical chaperon on endoplasmic reticulum (ER) stress and intrinsic apoptotic pathway. (a) iFECD was treated with or without TGF-β2 (10 ng/ml) for 24 hours, and 4 PBA (5 mM) was added to the medium to evaluate the effect of a chemical chaperone on unfolded protein formation. Unfolded protein was evaluated by aggresome staining and nuclei were stained with DAPI. Experiments were performed in triplicate. Scale bar: 50 µm. (b) Representative phase contrast images are shown. TGF-β2 induces cell detachment of iFECD, but 4 PBA suppressed the cell detachment. Experiments were performed in triplicate. Scale bar: 200 µm. (c) Effect of chemical chaperon on ER stress and apoptosis was evaluated. TGF-β2 treated samples were recovered after coincubation with 4 PBA and phosphorylation of PERK, expression of CHOP, and cleavage of caspase 3 were examined by western blotting. Full-length blot of caspase 3 is presented in Supplementary Fig. 1. GAPDH was used as internal control. Experiments were performed in triplicate. (d,e) Apoptotic cell were evaluated by Annexin V staining and evaluated by fluorescent microscope and flow cytometry. Nuclei were stained with DAPI for fluorescence microscopy analysis. Experiments were performed in triplicate. Scale bar: 50 µm. The statistical analysis was performed by Student’s t-test. *P < 0.01.
