Af3626

Paraffin-embedded stomach sections were dewaxed, rehydrated and antigen-retrieval was performed by heating in EDTA buffer (pH9) for 15 min in microwave pressure cooker. Section were incubated for 1 h at room temperature with goat anti-mouse IL-33 (1:150 dilution, R&D Systems, Cat# AF3626, RRID:AB_884269) and then for 1 h with AlexaFluor 568-conjugated donkey anti-goat secondary antibody (Molecular Probes Cat# A-11057, RRID:AB_142581). After counter staining with spectral DAPI (1:200 dilution, PerkinElmer, Cat# FP1490) for 5 min, sections were mounted with Vectashield mounting solution (VECTORlabs, Cat# H-1400). Imaging was preformed with a Vectra 3.0 Automated Quantitative Pathology Imaging System (PerkinElmer, Cat# CLS142338) and representative images were produced using Phenochart^TM v1.0.4 software (PerkinElmer).

Total protein extracts were prepared by homogenizing samples in lysis buffer (50 mmol/L Tris, 150 mmol/L NaCl, 2 mmol/L EDTA, 2 mmol/L EGTA, 0.2% Triton X-100, 0.3% NP-40, 0.1 mmol/L PMSF, 25 mmol/L NaF). For nuclear extracts, the NE-PER^TM nuclear and cytoplasmic extraction kit (Pierce, Thermofisher Scientific) was used according to the manufacturer’s instructions. Proteins were separated by SDS-PAGE under reducing conditions and then blotted onto nitrocellulose membranes. Membranes were blocked with 5% defatted milk in TBS-T (0.05 mol/L Tris, 0.15 mol/L NaCl, 0.05% Tween 20, pH 7.8). Thereafter, membranes were probed overnight at 4 °C with primary antibodies in the same blocking solution or 5% BSA in TBS-T and then incubated with secondary HRP-conjugated antibodies for 1 h at room temperature. Primary antibodies were: phospho-MLKL (1/500; ab196436; Abcam, Inc.); cleaved caspase-8 (1/1000, 8592, Cell Signaling Technology); p-cJUN (1/1000, 3270, Cell Signaling); cleaved IL-33 (1/1000; AF3626; R&D Systems), heme oxygenase-1 (HO-1, 1/2000, ADI-OSA-150-D, Enzo) and p65 (1/1000, 8242, Cell Signaling Technology). Total protein content for loading controls was assessed with Ponceau Red or by means of the fluorescence incorporated into tryptophan amino acids of proteins samples ran in Stain-Free^TM gels (BioRad).

Mice at denoted ages were administered one dose of vehicle (5% DMSO), imatinib (5 mg/kg; Sigma: SML1027) or SU16f (2 mg/kg; Tocris Bioscience: 3304) dissolved in 5% DMSO 1XPBS solution for 5 consecutive days via IP injection. Recombinant Pdgf-BB or IL-33 were administered to young mice at one dose of vehicle (1X PBS), Pdgf-BB (25 ng/mouse; PeproTech: 315-18), or IL-33 (12 µg/kg; PeproTech 210-33) for 5 consecutive days via IP injection. For anti-IL-33 neutralizing antibody experiments, TMX-induced 12-month-old mice were administered one dose of mouse IgG control (1 µg/mouse; Invitrogen 31903) or anti-IL-33 antibody (1 µg/mouse; R&D systems AF3626) by IP injection for 5 consecutive days. Subsequently, mice were cold temperature challenged. For fludarabine treatments, mice at denoted ages were randomized to one dose of vehicle (5% DMSO) or fludarabine (3 mg/kg; Tocris Bioscience: 3495) for 5 consecutive days via IP injection.

Endometriosis was induced in C57BL/6 mice as described above. All mice were treated with i.p. injections of exogenous recombinant mouse IL-33 at 1 μg per mouse (Biolegend, 580506) at the same dosing schedule as above; however, experimental mice were also in tandem treated i.p. with goat anti-mouse IL-33 antibody at 2.5 μg per mouse (R&D Systems, AF3626) and appropriate isotype control (R&D Systems, AB-1080C). The same processing for tissue harvest was conducted.

Control IgG and anti-IL-33 antibody (AF3626) were obtained from R&D Systems. For the in vivo experiments, the control antibody or the anti-IL-33 antibody were administered intraperitoneally once per week at a dose of 3 µg/mice starting 16 weeks after DMBA exposure [15 ]. The mice were euthanised 17 weeks after the first PMA cycle, and tumours were harvested for analysis.