Rsm 932a

RSM-932A (Cayman Chemical, Ann Arbor, MI) was reconstituted to 30 mg/mL in DMSO. For in vivo injection, RSM-932A was further diluted with DMSO and PBS to 1.5 mg/mL. Mice were injected intraperitoneally with 3 mg/kg of RSM-932A or 40% DMSO in PBS (vehicle group). In vivo injection experiments were conducted at least 3 independent times and as indicated in the figure legends.

BMDM were isolated and cultured as previously described [80 (link), 81 (link)]. Briefly, bone marrow cells were obtained from the femur and tibia by centrifugation [4 (link)]. Cells were differentiated into macrophages using 15–20% L929-conditioned media in complete DMEM (Wisent) containing 10% FBS (Wisent), 1% penicillin/streptomycin. Cells were plated into 15 cm dishes and allowed to differentiate for 6–8 days. Cells were lifted by gentle scraping in 10mM EDTA in PBS, counted, and seeded into culture plates for experiments at 1x10⁶/mL. Cells were treated for 24 h with DMSO as vehicle or inhibitors: hemicholinium-3 (Sigma-Aldrich), RSM-932a (Cayman Chemicals). Macrophages were polarized with 20 ng/mL recombinant IL-4 (Peprotech or Roche) or 100 ng/mL LPS (E. coli:B4, Sigma-Aldrich). Choline-free DMEM was formulated by preparing nutrient-deficient DMEM (US Biologicals, D9809) according to manufacturer’s instructions and supplementing with sodium pyruvate (Gibco), myo-inositol, L-methionine, and calcium D-pantothenate (Sigma-Aldrich).