Brefeldin a

Mouse splenic CD19⁺ B cells (1.5x10⁶/ml) were cultured in medium (RPMI 1640 glutamax; Thermo Fisher Scientific) containing 10% heat-inactivated fetal bovine serum (FBS; Gibco, Gaithersburg, MD, USA) and 100 μg/mL penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA) and restimulated for 2 days with SEA (20 μg/ml) to allow the detection of cytokines, as previously established for in vivo schistosome-exposed B cells [36 (link)]. Supernatants were collected for cytokine analysis by ELISA. Cells were cultured for an additional 4 hours with Brefeldin A (10 μg/ml; MedChemExpress) to detect intracellular IL-10 by flow cytometry. For in vitro Treg cell induction, SEA-stimulated CD19⁺ B cells were co-cultured with MACS-sorted CD4⁺CD25^- T cells at a 1:1 ratio (1 x 10⁶/ml each). After 4 days, the frequencies of Treg cells were determined by flow cytometry by gating on Foxp3⁺CD25⁺ cells in the CD3⁺CD4⁺ T cell population, and culture supernatants were collected for subsequent cytokine determination.

All the macrocycle compounds are commercially available. Brefeldin A was purchased from MedChemExpress, romidepsin and troleandomycin from Focus Biomolecules, simeprevir and paritaprevir from Invivochem, and pacritinib from Sigma Aldrich.

β-elemene (#63965) was purchased from Sigma-Aldrich. Gefitinib (#HY-50895), Rapamycin (#HY-10219), Brefeldin A (#HY-16592), 3-Methyladenine (#HY-19312), and Chloroquine (#HY-17589) were obtained from MedChemExpress. The Cell Counting Kit-8 (#MA0218), Phartmingen annexin V-FITC Apoptosis Detection Kit I (#556547), and Lyso-Tracker Red (#C1046) stain were obtained from Meilunbio, BD Biosciences, and Beyotime, respectively. The PrimeScript RT reagent kit (#RR037A) was purchased from Takara. The antibody against GAPDH (#5174), β-actin (#4970), LC3B (#3868), and SQSTM1 (#16177) were obtained from Cell Signaling Technology. The plasmids for overexpression of ATG5 (#RC210563) and ATG7 (#RC226545) were obtained from ORIGENE. EpiQuik m⁶A RNA Methylation Quantification kit (#710394) was purchased from EPIGENTEK.

The porcine fetal fibroblasts (PFFs) used in this study were obtained from Large White pigs at the Northeast Agricultural University Embryo Engineering Laboratory Experimental Pig Base. All animal-related procedures followed the guidelines set forth by the Institutional Animal Care and Use Committees (IACUCs) of Northeast Agriculture University. The PFF culture method has been previously published [23 (link)]. Briefly, PFFs were isolated from a 33-day-old foetus and cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco, New York, NY, USA) supplemented with 15% foetal bovine serum (FBS, HyClone, New York, NY, USA), 1% penicillin–streptomycin (Gibco, New York, NY, USA), 1% nonessential amino acids (Gibco, New York, NY, USA), and 2 mmol/L L-glutamine (Sigma, New York, NY, USA). The cells were passaged every two days using 0.25% trypsin-EDTA (Gibco, New York, NY, USA). The small molecules L-189 (CAS No. HY-15588), NU7441 (CAS No. HY-11006), SCR7 (CAS No. HY-12742), L755507 (CAS No. HY-19334), RS-1 (CAS No. HY-19793), and Brefeldin A (CAS No. HY-16592) were sourced from Med Chem Express Company (Monmouth Junction, NJ, USA). The small molecules were added in cell culture medium after CRISPR RNP electroporation at different concentrations.