2.1 Cell culture
Murine cone photoreceptor 661W cells were originally obtained from Dr. Muayyad Al-Ubaidi of the University of Oklahoma Health Sciences Center (Oklahoma, OK, USA) and cultured in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 IU/mL streptomycin (all from Hyclone, Beijing, China) in a humidified incubator with 5% CO2 and 95% air at 37°C. The cells were re-cultured with 0.05% trypsin-ethylene diaminetetra-acetic acid (EDTA) to detach the cells from the culture dishes.
2.2 Infection with lentiviral-mediated PARP short hairpin RNA (shRNA)
To knockdown PARP1 expression in cells, we purchased a lentivirus carrying PARP1 shRNA from GeneCopoeia (Rockville, MD, USA). A lentivirus carrying scrambled shRNA was used as a negative control (GeneCopoeia). The targeting PARP1 shRNA sequence was 5'-GAGTACATTGTCTACGACATT-3'; the negative control shRNA sequence was 5'-GCTTCGCGCCGTAGTCTTA-3'. The lentiviruses were produced by GeneCopoeia using a third-generation lentiviral package system (pRRE, pRSV-Rev, and pCMV-VSVG) in human embryonic kidney 293T (HEK-293T) cells. Lentivirus titer was assessed 72 h after cell infection. To knockdown PARP1 expression in 661W cells, we grew the cells and infected them with lentiviruses carrying PARP1 or negative control shRNA for 24 h. After confirmed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and western blot, stable cell lines were selected by 2-weeks’ treatment with 6 µg/mL hygromycin (Sangon Biotech; Shanghai, China).
2.3 Light exposure and drug treatment
Cells transfected with lentivirus and control cells were seeded into 6- or 96-well plates and grown to reach 70% confluency, then treated with a PI3K inhibitor (LY294002, Beyotime Biotechnology) that had previously been diluted in DMEM to a concentration of 33 µM or an Akt activator (SC79, Beyotime Biotechnology) that was previously diluted in DMEM to a concentration of 10 µg/ml. After 30 min of exposure to treatment, cells were exposed to the light (1,500 lux). The duration of exposure varied from 1 to 3 days, with all cells treated with the inhibitor or activator exposed to light for 1 day.
2.4 PI/Hoechst staining
Light-induced cell death was visualized with double nuclear staining with Hoechst dye and PI (both from Beyotime Biotechnology). Treated cells were stained with 2 µg/mL Hoechst dye at 37°C for 30 min and then with 5 µg/mL PI, before incubation in the dark at 4°C for 10 min. The stained cells were reviewed and scored under an inverted fluorescence Olympus microscope (Tokyo, Japan). The rate of cell death was calculated as: total number of PI-positive cells/total number of Hoechst-stained cells × 100%.
2.5 Animals and experiments
The animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the Second Hospital of Jilin University (Changchun, China) and followed the Guidelines of the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. Six-week-old male C57BL/6 J mice were purchased from Liaoning Changsheng Biotechnology (Cat. #211002300026556; Benxi, China) and housed under controlled temperature cycles (22 ± 1°C), with humidity ranging from 30–70% and an alternating 12-hour light/dark cycle, with ad libitum access to food and water.
For in vivo experiments, mice received oral treatment with a PARP inhibitor (ABT888, Beyotime Biotechnology) at 10 mg/kg per day for three consecutive days. Mice were housed in individual cages, and their pupils were dilated with atropine sulfate solution. Animals were then exposed to a bright continuous light for 12 h to induce retinal degeneration. The light source was a cool white 7500-lux fluorescent tube. Light intensity was measured with a portable lux meter (TES, Taiwan, China). Animals were then exposed to a normal light/dark cycle and received ABT888 treatment for another 4 days. Mice were then euthanized with an overdose of sodium pentobarbital. Eyes were immediately resected and subjected to morphologic analysis; one was allocated for histology, and the other was used for molecular analysis of the retina (see below for details).
2.6 Immunofluorescence
Cells were washed with phosphate-buffered saline (PBS) three times and fixed with 4% paraformaldehyde (Dingguo, Beijing, China) for 20 min, then treated with 0.2% Triton-X 100 (Dingguo). Frozen sections prepared from mouse retinal tissue were fixed in ice-cold acetone for 10 min and washed three times with PBS. Sections were then incubated with 3% bovine serum albumin (BSA) solution in PBS for 30 min. Cells and tissue samples were incubated at 4oC overnight in the following primary antibodies: monoclonal anti-mouse acetylated tubulin (1:200 dilution, Proteintech, Wuhan, China); anti-mouse Akt (phospho-Ser473; 1:100 dilution, Signalway Antibody, St. Louis, MO, USA). The next day, cells were washed with PBS three times, then incubated at room temperature for 30 min with the following secondary antibodies: Cy5-conjugated high-quality goat anti-mouse IgG (H + L) (1:200 dilution, GeneCopoeia, Rockville, MD, USA); goat anti-rabbit IgG (H + L)-conjugated with FITC (1:200 dilution, Proteintech). After three washes in PBS, cells were incubated at room temperature for 15 min with DAPI (Signalway Antibody) at a dilution of 1:100. After three PBS washes, the cells were examined under a fluorescence microscope (Olympus) or a confocal microscope (Olympus).
After stained for acetylated tubulin in cilia, all nuclei and their cilia were counted under a 400x microscopic field. Cells were counted in a total of 10 400x fields. Cell counts are presented as the percentages of cells with cilia in each 400x microscopic field.
2.7 Retinal histology
Mouse retina tissues were first fixed overnight in 4% paraformaldehyde at room temperature and dehydrated in a series of ethanol solutions, cleared in xylene, and then embedded in paraffin. Sagittal sections (thickness, 3 µm) of the retina were prepared and stained with hematoxylin and eosin (H&E). Images were captured under a microscope equipped with a charge-coupled device (CCD) camera (Tanon, Shanghai, China). We then measured the thickness of the outer nuclear layer (ONL) and the photoreceptors, with images obtained every 0.5 mm from the optic nerve head to the most peripheral region of the retina using the CellSens Entry program (Olympus, Tokyo, Japan). Average thickness values were calculated.
2.8 Transmission electron microscopy
Cells or retinal tissues were fixed in 3% glutaraldehyde (Huadong, Tianjin, China) at room temperature for 2–4 h and washed 4 times with PBS at 4oC, then further fixed in 1% osmium acid in 100 mM sodium dimethylarsone buffer at the room temperature for 2 h and washed with PBS for 5 min. Samples were then dehydrated in a series of ethanol solutions (10 min each) and submitted to centrifugation. Samples were then dehydrated in 90% acetone and pure acetone, three times, for 10 min each. Next, samples were incubated with acetone and epoxy resin embedding agent, then embedded in a capsule or plate with a newly prepared embedding agent (SPI, West Chester, PA, USA) at 35oC overnight. Samples were then stored at 60oC for 24–58 h. The ultrathin sections were prepared with an ultrathin slicer machine (JEOL, Japan) and stained with uranyl acetate and lead citrate, then reviewed under a transmission electron microscope (JEOL, Japan).
2.9 Western blot
Cultured cells were collected and sonicated in radioimmunoprecipitation assay buffer (RIPA; Beyotime Biotechnology). Fresh mouse retina tissue samples were submerged in liquid nitrogen and smashed using a hammer, then placed in 40 µL of RIPA buffer and sonicated. Samples were then centrifuged at 10,000 rpm for 15 min at 4°C for collection of the supernatants that were then subjected to the bicinchoninic acid assay to measure protein concentrations. For western blotting, 20 µg of cell lysate were dissolved in sample buffer and boiled for 5 min, then separated in 10% polyacrylamide gels containing 0.1% SDS and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were then blocked in 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween-20 (TBS-T) at room temperature for 1 h and incubated with the following primary antibodies: mouse polyclonal anti-β-actin (1:1000; Signalway Antibody); anti-PARP (1:1000; Cell Signaling Technology, Danvers, MA, USA); rabbit polyclonal anti-CEP164 (1:1000; Proteintech); rabbit polyclonal anti-ARL13B (1:1000; Proteintech); anti-p-PI3K (1:1000; Bioworld Technology, Minneapolis, MN, USA); anti-PI3-kinase p85α/γ (1:1000; Bioworld Technology); anti-Akt (phospho-Ser473; 1:1000; Signalway Antibody), rabbit monoclonal anti-Akt (pan; 1:1000; Cell Signaling Technology). Samples were incubated with a primary antibody for 3 h at the room temperature. Next, membranes were washed three times with TBS-T, then incubated with goat anti-mouse or goat anti-rabbit IgG secondary antibody-conjugated horseradish peroxidase (HRP) (1:3000 dilution for both; Signalway Antibody). The membranes were subsequently incubated with an enhanced chemiluminescence agent (Millipore, Billerica, MA, USA) and exposed to X-ray films.
2.10 Statistical analysis
Each experiment was performed in triplicate and repeated at least three times. Data were summarized as means ± SEM and statistically analyzed with SPSS v24.0 software (SPSS, Chicago, IL, USA). Differences between means were evaluated using one-way analysis of variance (ANOVA), followed by the post-hoc Bonferroni test. P < 0.05 was considered to be statistically significant.