Hpgds

At 24 h after debridement, protein samples of the wounded corneas were harvested. For each group, six corneas form six mice were collected. Samples (20 μg) were run on SDS-PAGE and then transferred to a polyvinylidene fluoride (PVDF) membrane. Membranes were blocked with 5% bovine serum albumin (BSA) at room temperature for 2 h and then incubated at 4°C overnight with the primary antibodies against hematopoietic prostaglandin D synthase (Hpgds) (Cayman, 160013), phosphorylated protein kinase B (p-AKT) (Cell Signaling Technology, 4060), AKT (Cell Signaling Technology, 4691), phosphorylated signal transducer and activator of transcription-3 (p-STAT3) (Abcam, ab76315), STAT3 (Cell Signaling Technology, 4904), and β-Actin (Affinity, AF7018), followed by incubation with horseradish peroxidase-conjugated secondary antibodies.

Purified human eosinophils or mouse eosinophils at 2 × 10⁶ cells/mL or 3 × 10⁶ cells/mL in Ca²⁺/Mg²⁺ HBSS (HBSS^+/+; pH 7.4) were pre-treated with the PI3K inhibitors wortmannin (1 μM; Biomol) and LY294002 (10 μM; Cayman Chemicals), PKC inhibitor calphostin C (1 μM; Biomol), pertussis toxin (PTX; 100 ng/mL), neutralizing monoclonal antibodies anti-CCL5 (10 μg/mL) and anti-CCR3 (10 μg/mL) (both from R&D), the PAF receptor antagonist BN52021 (10 μM), PGD₂ receptor antagonists BWA868c (200 nM; DP1 receptor) and Cay10471 (200 nM; DP2 receptor, or PGD₂ synthesis inhibitors HQL-79 (10 μM; H-PGDS) and AT-56 (10 μM; L-PGDS) (all from Cayman Chemicals) at 37°C for 30 min before stimulation with human recombinant (hr) or mouse recombinant (mr) leptin (0.5, 5, or 50 nM, as indicated; Peprotech) for 15 or 60 min in a water bath (37°C). Alternatively, eosinophils were also stimulated with PAF (1-O-hexadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine; 1 μM; Cayman Chemicals), hr CCL5 (also known as RANTES−100 ng/mL; R&D) or PGD₂ (25 nM; Cayman Chemicals). Each experimental condition was repeated at least three times with eosinophils purified from different donors or differentiated in vitro from different mouse bone marrows.