Animals were used in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research and all procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Augusta University. We used 8-week-old male wild-type (WT), 12/15-Lo knockout mice (12/15-Lo−/−; B6.129S2-Alox15tm1Fun/J; stock no: 002778, Jackson Laboratories, Bar Harbor, ME, USA), or Nox2-deficient mice (Nox2−/−; B6.129S-Cybbtm1Din/J; stock No: 002365, Jackson Laboratories) on C57BL/6J background. Diabetes was induced by intraperitoneal injection of streptozotocin (STZ) (Sigma, St Louis, MO, USA) at 50 mg/kg for 3–5 days until the mice developed diabetes (random blood glucose ≥13.9 mmol/l). Intravitreal injections were performed in non-diabetic mice as previously described using 12-HETE (Cayman Chemical, Ann Arbor, MI, USA) [25, 26].
Culture of HRECs
HRECs were purchased from Cell Systems Corporation (Kirkland, WA, USA) and were grown at 37°C under 5% CO2 in Endothelial Basal Medium-2 (EBM-2) (Lonza, Walkersville, MD, USA) supplemented with 10% FBS (vol./vol.) (Atlantic Biological, Norcross, GA, USA) and 1% penicillin/streptomycin (vol./vol.) (Corning, Tewksbury, MA, USA). These cells were negative for mycoplasma and the majority were >95% positive for von Willebrand factor/factor VIII and CD31 (endothelial markers), whereas the contaminating astrocytes (stained with glial fibrillary acidic protein [GFAP]) and pericytes (stained with neural/glial antigen 2 [NG2]) were insignificant <1%. After HRECs reached 90% confluence, the medium was changed to be serum free, followed by different treatment with 15-HETE (0.1 μmol/l, Cayman Chemical) or vehicle (ethanol) in the presence or absence of 4-phenylbutyric acid (PBA, 30 μmol/l, Santa Cruz, Dallas, TX, USA), VAS2870 (10 μmol/l, Sigma), or apocynin (30 μmol/l, Calbiochem, La Jolla, CA, USA). Of note, species-specific divergence between orthologous LO isoforms has been reported for mouse 12-LO, which is arachidonate 15-LO in humans. This difference implies that caution should be taken if experimental data on LO activity are being translated from one species to others [27, 28]. Therefore, 12-HETE has been used in all mouse experiments while 15-HETE has been used in all human retinal endothelial cell culture experiments. The doses of 12- and 15-HETE were selected according to what was previously detected in the vitreous of individuals with diabetic retinopathy, 50 ng/ml (approximately 0.1–0.2 μmol/l) .
Western blot analysis and immunoprecipitation
Western blot analysis was conducted on retinal or cell lysates using antibodies for XBP1s, XBP1, p-PERK, PERK, p-eIF2α, eIF2α, ATF4, ATF6, PDI, gp91phox, P47phox, vascular endothelial growth factor receptor-2 (VEGFR2), β-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ESM Table 1) according to the previously described method . To assess the phosphorylation of VEGFR2 in HRECs, immunoprecipitation was performed by the addition of an anti-VEGFR2 antibody and protein A agarose beads (Cell Signaling Technology, Beverly, MA, USA) per manufacturer’s instructions. This was followed by immunoblotting using anti-phosphotyrosine-PY20 antibody (BD Transduction Laboratories, San Diego, CA, USA).
Quantitative real-time RNA PCR
The mRNA levels of various ER stress markers were measured using TaqMan primers for Eif2a, Atf6, Chop (also known as Ddit3), Xbp1, Pdi (also known as Pdia3), calnexin and 18 s for mouse samples, or IRE1α, XBP1, PERK (also known as EIF2AK3), ATF4, ATF6, CHOP (also known as DDIT3), PDI (also known as PDIA2), BIP (also known as HSPA5) and calnexin for HREC samples (ESM Table 2) according to the previously described method .
Vascular endothelial growth factor (VEGF) level in HREC conditioned media was estimated using VEGF ELISA kits (R&D Systems, Minneapolis, MN, USA) per the manufacturer’s instructions.
In vitro leucocyte adhesion assay
This assay was done according to our previous procedure  where adherent leucocytes were counted under an inverted fluorescence microscope (excitation/emission = 480/520 nm).
Superoxide measurement using dihydroethidium
HRECs were incubated with dihydroethidium (DHE) (20 μmol/l, Invitrogen, Eugene, OR, USA; catalogue no. D11347) in Earle’s balanced salt solution (EBSS) for 30 min. The medium was then replaced by EBSS containing NADPH (100 μmol/l) as an enzyme substrate for NOX followed by 15-HETE treatment in the presence or absence of VAS2870 (NADPH oxidase inhibitor, 10 μmol/l, Sigma) or PBA (30 μmol/l, Santa Cruz). A microplate reader (BioTek Instruments, Winooski, VT, USA) was used to detect the fluorescence intensity at different time intervals.
Live imaging of intracellular calcium
Intracellular calcium (Ca2+) mobilisations were detected by the use of Cal-520, AM (AAT Bioquest, Sunnyvale, CA, USA; catalogue no. 21130) according to manufacturer’s instructions. Measurements were performed using a Zeiss 780 Inverted Confocal microscope (Zeiss 780 Inverted Confocal, Imaging Core Facility, Augusta University) at (excitation/emission = 490/525 nm).
Statistical data analysis
The results are expressed as means ± SEM. Differences among different experimental groups were assessed by one-way ANOVA or two-tailed t test. Results were considered significant for p < 0.05.