Anti gata 3

Following RA (Sigma-Aldrich, St. Louis, MO, USA) treatment or retroviral infection, cells in the exponential growth phase at 70–80% confluence were harvested at various time points and washed once with ice-cold phosphate-buffered saline. Cell pellets were suspended in SDS sample buffer and boiled for 10 min prior to centrifugation at 211 × g for 10 min. Samples were subjected to 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA). The membrane was probed with antibodies and binding was visualized using enhanced chemiluminescence (ECL; Beyotime Institute of Biotechnology, Haimen, China). The following primary antibodies were used: Rabbit polyclonal anti-GATA3 (1:100; H-48, sc-9009; Santa Cruz Biotechnology Inc., Dallas, TX, USA), mouse monoclonal anti-Mash1 (1:100; clone 24B72D11.1; BD Pharmingen, San Diego, CA, USA), rabbit polyclonal anti-peripherin (1:2,000; AB1530; Chemicon International, Inc., Billerica, MA, USA) and mouse monoclonal anti-α-tubulin (1:10,000; B-5-1-2; Sigma-Aldrich). Horseradish peroxidase-conjugated goat anti-mouse and goat anti-rabbit IgG (1:5,000, ICN, Bryan, OH, USA) were used as secondary antibodies.

The CO came from Brazil, obtained from the trunk of copaiba trees belonging to Copaifera langsdorffii. It was acquired from Pharmanostra (actual InfinityPharma) (São Paulo, SP, Brazil), lot 16E09-B027-009523. The chemical characterization of this tested CO was performed previously by GC-MS and GC-FID (Caputo et al., 2020 (link)).
OVA was from Sigma Aldrich^® (São Paulo, SP, Brazil), Ketamine (10%) from Syntec^® (São Paulo, SP, Brazil), and Xylazine (2%) from Ceva^® (São Paulo, SP, Brazil). The Star Trek Universal HRP Detection System kit (STUHRP700H, L10) was from Biocare Medical (Pacheco, CA, USA). The primary antibodies anti-GATA3, anti-STAT3, anti-TBET, and anti-FOXP3 were obtained from Santa Cruz Biotechnology (Dallas, Texas, USA), and the anti-IL-33 and the goat IgG hrp-conjugated antibody were from VWR International (Carnaxide, Portugal).

A total of 40 paired LUAD tissue specimens were collected in the Department of Respiratory Medicine, Taicang Affiliated Hospital of Soochow University. All participants were provided with written informed consent at the time of recruitment. All samples were kept at -80°C for storage. All cases had clinically and pathologically confirmed diagnoses of LUAD based on the Revised International System for Staging Lung Cancer. Ethics approval was obtained from the local Institutional Review Board committee. Paraffin-embedded tissues were sectioned into 5-μm slides and mounted. Slides were deparaffinized, rehydrated with ethanol, and quenched in 30% vol/vol hydrogen peroxide/methanol (1:9) for 15 min. Non-specific antigens were blocked by incubating in 2% BSA and 0.1% Triton X-100 for 30 min at room temperature. The sections were incubated with primary antibodies anti-MTFR2 (1:100 dilution; Sigma-Aldrich, St. Louis, Missouri, USA) and anti-GATA3 (1:100 dilution, Santa Cruz, Dallas, Texas, USA) overnight, followed by a secondary antibody, then stained with diaminobenzidine (DAB, Vector Laboratories, Burlingame, CA) and hematoxylin (nuclei stain). ImageScope software was used to quantify the intensity and positively stained areas. Two pathologists independently scored immunohistochemistry staining.