E7781

Cells (5 × 10³) were plated on gelatin (1% in PBS)-coated 96-well plates (130188; Thermo Fisher). The next day, cells were incubated with different concentrations of Erastin (0.05–10 µM, E7781, Sigma) for 24 h. To assess the effect of ERα antagonism, 3.0 × 10³ cells were incubated with different concentrations of methyl-piperidino-pyrazole (MPP, M7068; Sigma) for 24 h and then further incubated with Erastin for another 24 h. The viability of the cells was assessed by MTS assay (G5421; Promega, Madison, WI, USA) by measuring absorbance at 490 nm using a plate reader (Epoch, BioTek Instruments, Winooski, VT, USA). Samples were prepared in quadruplicate and repeated twice.

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.

Apoptosis was induced using camptothecin (10 μM), cycloheximide (200 μM), etoposide (100 μM) (ab102480, Abcam), or anti-FAS antibody (305702, 150 ng/ml, BioLegend). Ferroptosis and necroptosis were induced after treatment with selective activators, Erastin (E7781, 1 μM, Sigma–Aldrich) or L-Buthionine-sulfoximine (BSO) (B2515, 1 μM, Sigma–Aldrich) for ferroptosis and Emodin (E7881, 5 μM, Sigma–Aldrich) or Shikonin (S7576, 1 μM, Sigma–Aldrich) for necroptosis. After fixation, the cells were treated with serum-free protein blocking solution (DAKO) and incubated with anti-PIP₃ IgM antibody (Z-P345, 1:100, Echelon Biosciences), CD14, AKT PHD, AKT-PHD/eGFP, or Annexin V. For immunofluorescence studies, tissue sections (2.5 μm) were blocked (DAKO) and incubated with anti-PI(3,4,5)P₃ and anti-cleaved caspase 3 antibodies. After mounting, the sections were imaged with an LSM 700 laser-scanning confocal microscope (Carl Zeiss), and images were analyzed with ZEN 2010 Software (Carl Zeiss).