Ml 265

PBMCs were isolated using density gradient centrifugation using Lymphoprep (STEMCELL Technologies). Monocytes isolated by plastic adherence were differentiated into macrophages in RPMI 1640 medium (Life Technologies) supplemented with 20 ng/ml of M-CSF (eBioscience) and 10% of FBS (Lonza) for 5 d. Alternatively, monocytes were isolated using the EasySep human monocyte enrichment kit without CD16 depletion (STEMCELL Technologies). Macrophages were further differentiated into M1 or M2 macrophages by stimulation with 100 U/ml of IFN-γ (Sino Biologicals) and 100 ng/ml of LPS (Sigma-Aldrich), or 10 ng/ml of IL-4 and 10 ng/ml of IL-13 (Sino Biologicals). Attached macrophages were dissociated from plates using StemPro Accutase Cell Dissociation Reagent (Life Technologies).
To scavenge ROS, macrophages were stimulated in the presence of the ROS-scavenger Tempol (Sigma-Aldrich) or the mtROS scavenger MitoTempo (Santa Cruz Biotechnology, Inc.). Assembly of the NOX2 membrane complex was inhibited with gp91dstat (Anaspec). Glycolytic activity was blocked with 10 mM of 2-DG or by using glucose free RPMI 1640 medium (Life Technologies). PKM2 tetramerization was enforced with 50 µM of ML265 (Cayman Chemical). HIF-1α was inhibited with 10 µM of CAS (934593–90-5; Santa Cruz Biotechnology, Inc.). STAT3 phosphorylation was inhibited with 5 µM of Stattic (Sigma-Aldrich).

Adipocyte differentiation was initiated by culturing confluent cells in adipogenic induction medium consisting of the growth medium supplemented with 160 nM insulin (Sigma-Aldrich, I9278), 250 nM dexamethasone (Sigma-Aldrich, D4902), and 0.5 mM 1-methyl-3-isobutylxanthine (IBMX, Sigma-Aldrich, I7018) (day 0). Forty-eight hours later (day 2), the cells were switched to maintenance medium supplemented just with insulin. After 48 hours (day 4), the cells were placed back in unsupplemented maintenance medium, which was changed every 48 hours. For lysosomal flux assays, cells were treated with 20 mM ammonium chloride (Sigma-Aldrich, AX12701) or 100 μM leupeptin (Thermo Fisher Scientific, BP266225) for 12 hours at the indicated time points in differentiation. For proteasomal flux assays, cells were treated with lactacystin (5 μM; Enzo Life Sciences, BMLPI104) for 12 hours at the indicated time points, and efficacy of inhibition was monitored by tracking changes in K48-ubiquitinated proteins. TGFβ inhibitor (SB525334, Tocris, 3211) was added to differentiating cells at day 1.5 at 1 μM for 12 hours. ML265 (Cayman Chemical, 13942) was added to activate glycolysis in control cells at 1 and 2 μM for 12 hours.