Senexin a

Lymphocytes were isolated from peripheral lymph nodes and spleen of age- and sex- matched mice and purified with CD4 microbeads (L3T4, Miltenyi Biotec). Purified CD4⁺ T cells were cultured in RPMI 1640 medium containing 10% FBS, 1% penicillin-streptomycin and 75 μM of β-Mercaptoethanol and activated with plate-coated 2.5 μg/ml CD3 (145-2c11, Bio-X-Cell) and 1 μg/ml CD28 (37.51, Bio-X-Cell) antibodies. For Treg differentiation, designated doses of TGF-β (0.01–2 ng/ml) and IL-2 (20 U/ml) were added into culture medium. CCT251921 (HY-19984, MedChem Express) and Senexin A (487510, Tocris Bioscience) were used to block Cdk8/Cdk19 activity. TGF-β receptor I (Alk5) inhibitor SB431542 (S1067, Selleckchem) was used to block TGF-β signaling. For proliferation assay, 2 μM of CFSE (C1157, Life Technologies) was used to label CD4⁺ T cells before differentiation.

Salivary glands were dissected and mounted as described (Gomez-Lamarca et al., 2018 (link)) by submerging mid L3 larvae in M3 Shields and Sang media (Sigma-Aldrich, S3652) supplemented with 5% fetal bovine serum (Sigma-Aldrich, F9665) and 1× Antibiotic-Antimycotic (Gibco, 15240-062). For drug and hormonal treatments, dissected glands were incubated for an hour with the following compounds: triptolide (10 µM, Sigma-Aldrich T3652), ecdysone (5 µM, Cayman Chemicals 16145), A485 (5 µM, Cayman Chemicals 24119), Senexin A (1 µM, Tocris 4875), and Senexin B (2 µM, Cayman Chemicals 24119). After dissection and any treatments glands were mounted into polylysine-coated coverslips in dissection media supplemented with methyl-cellulose (Sigma-Aldrich, M0387-100G).