Cell culture and treatments
The human RPE cell line ARPE-19 (CRL-2302, ATCC, USA) and human CECs (HCECs, CP-H092, Procell, China) were grown in Dulbecco’s modified Eagle medium/Ham’s F12 medium (DMEM/F-12; 8118247, Gibco, USA) supplemented with 10% fetal bovine serum (FBS; 10099141, Gibco) and antibiotic-antimycotic solution (15240062, Gibco). ARPE-19 cells and HCECs cultured in 95% air and 5% CO2 for 24 h were taken as the normal (normoxia) groups. Cells cultured in 1% O2, 5% CO2 and 94% N2 in an oxygen-controlled chamber for 24 h were taken as the hypoxia groups. HCECs were treated with human recombinant ANXA1 protein (3770-AN, R&D Systems, USA; 100 nM for 24 h), WRW4 (FPR2 antagonist; 2262, Tocris, USA; 10 μM for 24 h), SHP099 (SHP2 inhibitor; S8278, Selleck, USA; 0.1 μM for 24 h), ARPE-19 cell conditioned culture medium (CCM; hypoxic culture for 24 h), ANXA1 neutralizing antibodies (ab46686, Abcam, USA; 1 μM for 24 h), adenosine triphosphate (ATP; NLRP3 inflammasome agonist; 10988537001, Roche, USA; 5 mM for 24 h) or a caspase-1 CRISPR activation plasmid (sc-417320-ACT, Santa Cruz Biotechnology, USA; 1 μg for 24 h).
The cells were cultured to 70% confluence, and subjected to the designated treatments. The cells were lysed at 4°C using radioimmunoprecipitation assay (RIPA) lysis buffer (R0278, Sigma Aldrich, USA). Protein concentrations were determined using a BCA protein assay kit (A53225, Thermo Fisher Scientific, USA). Samples (80 µg protein) were resolved by 4-20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; WXP81612BOX, Invitrogen, USA), transferred to polyvinylidene difluoride (PVDF) membranes (IPVH00010, Millipore, USA), and incubated with one of the following antibodies: ANXA1 (21990-1-AP, Proteintech, USA), FPR2 (PA5-75750, Invitrogen), p-SHP2 (Tyr542; #3751, Cell Signaling Technology), SHP2 (#3752, Cell Signaling Technology), ASC (67494-1-Ig, Proteintech), NLRP3 (19771-1-AP, Proteintech), IL-1β (including pro-IL-1β and cleaved-IL-1β; AF-401, R&D Systems), N-GSDMD (ab215203, Abcam), GSDMD (20770-1-AP, Proteintech) and caspase-1 (22915-1-AP, Proteintech). The membranes were washed three times with phosphate-buffered saline with Tween detergent (PBST) and incubated with horseradish peroxidase (HRP)-conjugated goat-anti-rabbit (SA00001-2, Proteintech; 1:5000) or HRP-conjugated goat-anti-mouse antibodies (SA00001-1, Proteintech; 1:5000) at room temperature for 2 h. GAPDH (600004-1-IG, Proteintech; 1:50000) and caveolin 1 (Cav1; 16447-1-AP, Proteintech) were used as loading controls. Unless otherwise indicated, the dilution for each antibody was 1:1000. Following an additional 10 min of washing with PBST, the protein bands were visualized using an electrochemiluminescence (ECL) assay kit (JP001B250, CLINX, China). The density of each band was measured using ImageJ software. The expression level of each target protein was normalized to the GAPDH or Cav1 protein expression level and compared with that of the normal group, which was assigned a value of 1.
Enzyme-linked immunosorbent assay (ELISA)
The levels of ANXA1 were measured using a human ANXA1 ELISA kit (NBP2-60538, Novus Biologicals, USA) according to the manufacturer’s instructions. The detection range of the kit was 0.313-20 ng/ml. The absorbance was measured using a microplate reader (Bio-Rad Laboratories, USA) at a wavelength of 450 nm and a correction at 655 nm.
Plasma membrane and cytosol isolation
Plasma membrane and cytosolic fractions were isolated from HCECs using a plasma membrane protein extraction kit (ab65400, Abcam, USA).
Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay
HCECs were seeded on glass coverslips in 24-well plates. After different treatments for 24 h, the TUNEL assay was done using a in situ cell death detection kit (11684795910, Roche), and the cells were observed under a TCS-SP2 confocal microscope (Leica Microsystems, Germany).
5-Ethynyl-2′-deoxyuridine (EdU) incorporation assay
Cell proliferation was analyzed using the EdU Apollo 488 assay kit (C10310-1, RiboBio, China). The images were viewed using a confocal microscope at 100 × magnification. The number of stained nuclei was counted and used to determine the percentage of the total number of nuclei in each image.
Wound healing assay
Confluent monolayers of HCECs in 6-well plates were scratched with pipet tips, leading to one acellular 0.5-mm-wide lane per well. After the cells were washed twice, the HCECs were exposed to different treatments. Photographs were taken after 24 h of incubation. The amount of migration was determined by ImageJ software.
Tube formation assay
A 24-well culture plate was coated with 100 μl of 10 mg/ml precooled growth factor-reduced Matrigel (354230, Corning, USA). HCECs were seeded on Matrigel-coated plates at concentrations of 5 × 104 cells per well and exposed to different treatments. After 24 h of culture, images were acquired using an optical microscope (× 100 magnification). The average length of the tube branches in four areas of each sample was assessed under an inverted light microscope.
Mouse laser-induced CNV model
After anesthesia and pupil dilation, male C57BL/6J mice (purchased from the Laboratory Animal Center of Soochow University) (aged 10 weeks) were subjected to laser photocoagulation using a PASCAL diode ophthalmic laser system (neodymium-doped yttrium aluminum garnet [Nd:YAG], 532 nm; Topcon Medical Laser Systems, Santa Clara, USA) with the following parameters including 200 mm spot size, 0.02 s duration, and 100 mW power. Four laser spots were made around the optic nerve heads of both eyes in each mouse to induce CNV. The rupture of Bruch’s membrane was verified by observing a bubble at each laser spot. Approval was obtained from the Animal Research Ethics Committee of Soochow University in agreement with the Chinese National Standard.
The mice were allocated into the normal, CNV 7 d, CNV 7 d + PBS, CNV 7 d + ANXA1 neutralizing antibody (intravitreal injection of 2 μl of 50 μg/ml antibody on day 3), CNV 7 d + WRW4 (intraperitoneal injection; 1 μg/kg/d from day 0 to day 6), CNV 7 d + SHP099 (oral gavage, 50 mg/kg/d from day 0 to day 6), CNV 7 d + ATP (intravitreal injection; 5 mM on day 3), and CNV + caspase-1 plasmid (intravitreal injection; 1 μg on day 3) groups. There were five mice in each group. Three mice were excluded due to hemorrhage at the site of laser administration.
Immunohistochemical analysis of RPE-choroid cryosections
Immunohistochemical analysis of ANXA1, FPR2, p-SHP2, NLRP3 or caspase-1 was performed on flattened retina-RPE-choroid complexes 7 d after laser exposure as previously described . The antibodies used immunohistochemical analysis included anti-ANXA1 (PA5-27315, Invitrogen), anti-FPR2 (NLS1878, Novus Biologicals, USA), anti-p-SHP2 (STJ90741, St. John’s Laboratory, USA), anti-NLRP3 (MA5-32255, Invitrogen), and anti-caspase-1 (MA5-32909, Invitrogen).
Immunofluorescence analysis of mouse choroidal flat mounts
The mouse choroidal flat mounts were prepared as previously described . The antibodies and reagents used included Alexa Fluor™ 488-conjugated anti-isolectin B4 (IB4) from Griffonia simplicifolia (I21411, Invitrogen), Alexa Fluor™ 594-conjugated anti-collagen IV (ITT5767, G-Biosciences, USA) and 4’, 6-diamidino-2-phenylindole (DAPI; D9542, Sigma Aldrich).
All results are represented as the means ± SEM. Statistical significance between groups was evaluated with Student’s unpaired t-tests (two-tailed). A value of P < 0.05 was considered to be statistically significant.