213 ilb

After proliferation submerged culture condition, the first-passage HAECs were seeded in 12-well Transwell plates as previously described (39 (link), 45 (link)). When confluent, HAECs were shifted to ALI culture with 50 μL apical media for 8 days to achieve full differentiation with and without IL-13 (10 ng/mL) stimulation (R&D Systems, 213-ILB).
Dicer siRNA KD of ALOX15 was performed in 12-well Transwell plates, as previously described (45 (link)). After transfection for 24 hours, IL-13 was added into lower chamber media every 48 hours for up to 7 days. ALOX15 dicer siRNA was purchased from IDT.
A selective enzymatic inhibitor of 15LO1, BLX2477 (gift from Hans-Erik Claesson, Karolinska Institutet, Stockholm, Sweden; ref. 15 (link)), was added at a final concentration of 2 mM for 24 hours. A selective enzyme inhibitor of SLC7A11, erastin (Sigma-Aldrich, E7781; ref. 19 ) was added at 10 μM into the medium for 24 hours before harvest, with dimethyl sulfoxide or water added as vehicle control. Total protein from freshly brushed and cultured HAECs was collected in cell lysis buffer, homogenized by sonication, and then centrifuged at 1,699g for 10 minutes. The apical supernatant was used for GSH:GSSG and protein analysis.

M2 and M1 macrophages were generated by the generally established methods [38 (link),39 (link)]. Human peripheral blood mononuclear cells (PBMCs) were collected from the peripheral blood of healthy volunteers using a BD Vacutainer^® CPT™ (BD Bioscience), and monocytes were isolated using a MidiMACS™ Separator (130-042-302, Miltenybiotec, Bergisch Gladbach, Germany), LS column (130-042-401, Miltenybiotec) and CD14 MicroBeads human (130-050-201, Miltenybiotec). CD14⁺ monocytes were cultured in a 12-well plate at a density of 5 × 10⁵ per well at 37 °C in 5% CO₂ atmosphere in RPMI1640 (Sigma-Aldrich) supplemented with 20 ng/mL of M-CSF (216-MC, R&D systems, Minneapolis, MN, USA), penicillin/streptomycin and 10% heat-inactivated FBS for 6 days. Subsequently, we continued to culture the cells in the same medium for 3 days to generate M0 macrophages, while we added 20 ng/mL each of IL-4 (204-IL, R&D Systems), IL-10 (217-IL, R&D systems) and IL-13 (213-ILB, R&D systems) into the culture medium and continued to culture the cells for 3 days for the generation of M2 macrophages. To differentiate M1 macrophages, we utilized 20 ng/mL of GM-CSF (7954-GM, R&D Systems) and 20 ng/mL each of LPS (L2630, Sigma-Aldrich), IFN-γ (285-IF, R&D Systems) and IL-6 (206-IL, R&D Systems).

THP-1 cells were differentiated into macrophages induced by 150 nM phorbol 12-myristate 13-acetate (PMA; Sigma, USA) with 24 h. Then, macrophage M2 polarization was obtained by incubation with 20 ng/ml of interleukin 4 (R&D Systems, #204-IL) and 20 ng/ml of interleukin 13 (R&D Systems, #213-ILB) for 48 h. M2 macrophage infiltration assays were applied through seeding 1.0×10⁵ M2 macrophage cells (300μl) without serum in the upper chamber of a Transwell plate for 48 h (size 5mm, Corning, NY, USA). In bottom plate, U87 and U251 glioma cells (1.0×10⁵) were cultured with10% FBS in DMEM (700μl). After incubation for 48 h, the cells in the upper chamber were fixed with 4% paraformaldehyde for 10 min and stained with 0.1% crystal violet for 10s. The infiltrated M2 macrophage cells were counted in three randomly selected fields from each membrane.

THP-1 (Human acute monocytic leukemia cell line) cells and L02 cells were purchased from ATCC. The cells were all cultured in RPMI 1640 containing 10% heat-inactivated FBS and 1% penicillin–streptomycin in a 5% CO₂ atmosphere at 37 °C. THP-1 monocytes were differentiated into M0-like macrophages by incubation with 160 ng/ml phorbol 12-myristate 13-acetate (PMA, Sigma, P8139) for 48 h in RPMI medium. PMA-induced macrophages were polarized into M1 macrophages by incubation with 20 ng/ml of IFN-γ (R&D system, #285-IF) and 10 pg/ml of LPS (Sigma, #8630). Macrophage M2 polarization was obtained by incubation with 20 ng/ml of interleukin 4 (R&D system, #204-IL) and interleukin 13 (R&D system, #213-ILB). In the co-culture experiments, 0.5 × 10⁶ THP-1 monocytes were cultivated and differentiated for 48 h before being incubated respectively with 0.5 × 10⁶ L02-SCR or L02-Sh cells in 6-well Transwell plates for 48 h^{37 (link)} .

Human umbilical vein endothelial cells (HUVEC) were purchased from PromoCell (Heidelberg, Germany), and cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Carlsbad, CA, USA) with 10% (v/v) fetal bovine serum (FBS) (Gibco) and antibiotics (100 IU/mL penicillin and 100 mg/mL streptomycin) (Gibco). The A549 cell line was purchased from Cell Resource Center of Life Sciences (Shanghai, China), and cultured in Roswell Park Memorial Institute 1640 medium (RPMI 1640) (Gibco) containing 10% FBS (Gibco). HUVEC were treated with recombinant human IL-13 protein (50 ng/mL) (213-ILB, R&D systems, Minneapolis, MN, USA) and STAT6 inhibitor, AS1517499 (AS) (1 mM) (HY-100614, MCE, NJ, USA).