May grunwald

13C-metabolic labeling was performed following published methods (47 (link)). Kelly were plated at 5 × 10⁵/well in 6-well plates in RPMI/10% FBS overnight before medium was changed to MEM (Sigma-Aldrich #51416C) containing 10% FBS, NEAA, 2g/L glucose and 2mM 50% 13C-uniformly labeled glutamine. For 13C glucose labeling, the same medium was used except that the glucose component was 50% 13C-uniformly labeled glucose (Sigma-Aldrich) and glutamine was unlabelled (2mM). Inhibitor or vehicle was added to cells for 6h or 24h before harvesting for media and polar metabolite fractions (47 (link)). GC-MS metabolomic analyses were performed as published (48 (link)) on polar metabolite or media fractions. Media was also analysed for glucose and lactate using a YSI model 7100 enzyme analyzer.
For real-time metabolic analyses, 80,000 Kelly cells were plated in wells of a XF24 plate (Seahorse Bioscience), and next day media was replaced with base media containing 2g/L glucose and 200 mM glutamine and analysed on a Seahorse XF24-3 Bioanalyser. After determination of baseline ECAR and OCR, 1μM of SR13800 or vehicle was injected onto cells. Results were normalized by cell count in five fields of methanol-fixed cells stained with May-Grunwald (Sigma-Aldrich) and Giemsa (Sigma-Aldrich).

Methanol, glacial acetic acid, microscope slides, sodium citrate, sodium chloride, potassium chloride (0.05 M KCl), Giesma stain, May-Grunwald, and heparin were purchased from Sigma-Aldrich (St. Louis, MO, USA). DCFH-DA (2’, 7-dichlorofluorescin diacetate) assay kit was obtained from Molecular Probes, Inc. USA. Comet assay kit was purchased from Trevigen, Inc. (Gaithersburg MD, USA. All reagents were of analytical grade or highest grade available.

Anti-mouse F4/80 Ab (Cl:A3-1) and anti-TGF-β (AHP1734) was purchased from Serotec (Raleigh, NC). Anti-p50 (ab7971), anti-p65/RelA (ab7970), anti-iNOS (ab3523), and anti- TNF-α (ab 6671) were purchased from Abcam (Cambridge, Massachusetts). F4/80-FITC-conjugate (BM8), CD36-PE-conjugate (No.72-1) were purchased from eBioscience (San Diego, California). Anti- IL-1β Ab (AF-401-NA), recombinant mouse (rm) GM-CSF (rm GM-CSF), rm IL-4, and Duoset TNF-α ELISA kit were purchased from R&D Systems (Minneapolis, MN). Anti-phospho p65/RelA (3033) was purchased from Cell Signaling (Danvers, MA). Anti-Arg1 (sc-18354) was purchased from Santa Cruz Biotechnology Inc. (Dallas, TX). Vectastain ABC kit for immunohistochemistry and Vectashield mounting medium was purchased from Vector Laboratories (Burlingame, CA). May-Grunwald and Wright-Giemsa stains were obtained from Sigma (St. Louis, Missouri). Filters (0.45-μm pore size, mixed cellulose ester) were acquired from Millipore (Billerica, MA) and PDMS from Invotec International (Jacksonville, FL). PDMS disks (4 mm in diameter and 1 mm thick) were cut with the aid of a biopsy punch (Acuderm; Fort Lauderdale, FL).