ARPE-19 cells (American Type Culture Collection, Manassas, VA, USA) at passages 12, absent of endogenous A2E were cultivated under 37 °C humidified 5% CO2 circumstance in Dulbecco’s modified Eagle’s/ Ham’s F12 medium (DMEM/F12; Invitrogen, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin, as previously described . The RPEs were delivered in different culture plates based on each experiment’s requirement. When achieving confluence, RPEs were transferred to serum-free medium for another 24 h before accepting treatments.
A2E synthesis and treatment paradigm
A2E was prepared from 100 mg all-trans-retinal (Sigma Aldrich, St. Louis, MO, USA) and 9.5 mg ethanolamine in 2 ml ethanol as previously described . A2E was stored in dimethyl sulfoxide (DMSO) at 25 mM in the dark under − 80 °C.
Confluent RPEs were cultivated with 25 μM A2E in medium for 2 h. Then, extracellular A2E was washed off. After A2E intaking, RPEs were illuminated by 460 ± 20 nm wavelength light (4000 lx; OSRAM GmbH, Augsburg, Germany) for 20 min, as previously described . To regulate autophagy, RPEs were pretreated with 5 mM 3-methyladenine (3-MA) or 10 nM rapamycin for 2 h before A2E loading and illumination.
Western blot analysis
RPEs grown in 90-mm diameter dishes were harvested in lysis buffer after indicated treatments. After centrifugation at 4 °C, supernatants containing proteins were collected and reserved at − 80 °C until use. A Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA) was used for protein immunoblot analysis. The protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After transfer of proteins, membranes were blocked and incubated overnight at 4 °C with the primary antibodies including GRP78 monoclonal antibody (1:1000; Epitomics Inc., Burlingame, CA, USA), CHOP polyclonal antibody (1:200; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), caspase-12 polyclonal antibody (1:200; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), LC3 monoclonal antibody (1:1000; Cell Signaling Technology, Danvers, MA, USA), AMPK monoclonal antibody (1:1000; Cell Signaling Technology, Danvers, MA, USA), phospho-AMPK monoclonal antibody (1:1000; Cell Signaling Technology, Danvers, MA, USA), mTOR monoclonal antibody (1:1000; Cell Signaling Technology, Danvers, MA, USA), or phospho-mTOR monoclonal antibody (1:1000; Cell Signaling Technology, Danvers, MA, USA). Secondary antibody conjugated with horseradish peroxidase (IgG-HRP; 1:2000; Cell Signaling Technology, Danvers, MA, USA) and enhanced chemiluminescence kit (ECL; GE Healthcare Life Sciences, Buckinghamshire, UK) were used to detect binding.
Transmission electron microscopy analysis
The treated RPEs were washed and fixed in 0.1 M cacodylate buffer (pH = 7.4) containing 2.5% glutaraldehyde and 2% paraformaldehyde for 1 h at room temperature. The samples were fixed for 1 h in 1% osmium tetroxide, prior to dehydration in a graded ethanol series and embedded in epon 812 resin. Then, ultra-thin sections containing cells were block-stained with uranyl acetate and lead citrate; and examined with a transmission electron microscope (CM120; Philips, Eindhoven, Netherlands). We evaluated a minimum of 6 images per sample.
After completion of respective treatments, RPEs were washed with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde for 20 min. The cells were then permeabilized with 0.1% Triton X-100 for 15 min and blocked for 1 h at room temperature. The cells were subsequently incubated with primary LC3 antibody in blocking solution at 4 °C overnight. The next day, cells were washed and then incubated with an Alexa Fluor 555-conjugated secondary antibody (IgG; 1:1000, Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature. The cells were briefly stained with 4′,6′-diamidino-2-phenylindole (DAPI) to visualize the nuclei. The fluorescence of LC3 punctate was detected by a confocal laser microscope (FV1000; Olympus, Japan) with a 540 nm band-pass filter after excitation at 488 nm. DAPI was excited at 568 nm and visualized with 670 nm band-pass filters. Autophagosomes were quantified by counting the number of LC3 fluorescent puncta in per RPE cell.
Short hairpin RNA interference of GRP78
Lentiviral vectors carrying a short hairpin RNA (shRNA) targeting GRP78 or negative control shRNA were produced by GenePharma Co. Ltd. (Shanghai, China). The sequence of shRNA targeting GRP78 was 5′-GGAACTGGAAGAAATTGTTCA-3′. The sequence of shRNA used as a negative control was 5′-TTCTCCGAACGTGTCACGT-3′. Stably transfected RPEs were selected and obtained as previously described .
After the indicated treatments, RPEs were washed thrice with PBS and fixed for 10 min with 4% paraformaldehyde at room temperature. After fixation, TUNEL staining was performed for 30 min at 37 °C in the dark. Then, cells were washed with PBS three times and examined under a confocal laser microscope. The number of death cells, which were identified as having TUNEL positive nuclei, in RPEs was counted.
These assays were performed at least three times. All quantitative data was recorded as mean ± SD. SPSS 21.0 version (IBM SPSS, Chicago, IL, USA) was used to evaluate difference by Student’s t-test or one-way analysis of variance (ANOVA). A p-value below 0.05 was considered statistically significant.