Characterization of Cellular Morphological Changes

To monitor the effect of DR on cellular morphological changes, cells were treated with DR for 12 hours, and images were captured under a phase-contrast microscope. To examine the morphology of endoplasmic reticulum (ER), cells were visualized by staining with specific fluorescent tracker dye according to the instructions of the manufacturer with slight modification. Briefly, after treatment with DR, cells were incubated with ER-Tracker Blue-White DPX for 30 minutes at 37°C. Cells were then washed with phosphate buffered saline (PBS) and visualized under a fluorescence microscope. Furthermore, to characterize the morphology of ER, cells were cultured on sterile poly-l-lysine coated glass cover slips for approximately 50% to 60% confluent condition in DMEM. For staining of ER membrane protein calnexin, cells were fixed using 4% paraformaldehyde and permeabilized with 0.2% Triton-X 100 in PBS for 10 minutes followed by PBS washes. After blocking with 2% bovine serum albumin (BSA), cells were incubated with rabbit anti-calnexin antibody (SC-11397) in T-TBS (100 mM Tris-HCl, pH 7.5; 150 mM NaCl and 0.05% Tween-20) containing 2% BSA for overnight at 4°C. After T-TBS washes, cells were incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG for 1 hour in the dark. The cover slips were washed with T-TBS, mounted with a drop of mounting medium and sealed with nail polish to prevent drying and movement under fluorescence microscope.¹³