Wild type (WT) and Aconitate decarboxylase 1 knockout (Acod1-/-) BMDM treated with Imiquimod for 12h

The Krebs cycle enzyme Aconitate Decarboxylase 1 (ACOD1) mediates itaconate synthesis in monocytes and macrophages. Here, we explore the role of endogenous ACOD1-itaconate pathway in the activation of BMDM after imiquimod treatment. Female wild type (WT) mice and ACOD1 knockout mice (Acod1-/-) were purchased from Jackson Laboratory (strains JX5304 and JX29340, respectively). Mouse bone marrow cells were flushed out with cold RPMI 1640 medium (Gibco) containing 1% penicillin/streptomycin and were then centrifuged, washed with PBS 1X (Gibco) and filtered through a 70-mm filter mesh. Bone marrow cells were counted and cultured at 1.0 x 106 cells/mL in Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher Scientific), supplemented with 10% fetal bovine serum (FBS, Sigma Aldrich), 1% penicillin/streptomycin, and M-CSF 25 ng/mL (R&D). M-CSF was replaced every 2 days. RNA was isolated from BMDM exposed or not, to IMQ for 12 hours. Tri Reagent (Sigma-Aldrich) was used followed by column-based purification with RNA Clean & Concentrator-5 Kit (Zymo Research).