Anti cd36 pe

For the in culture differentiation assay, cells were transfected with mTOG or SCR oligos on days 0 and 7 of the protocol. The differentiation assay was performed as previously described^{34 (link)}. Briefly, the cells were expanded for 7 d in stem cell medium (IMDM (Sigma), 15% BIT (StemCell Technologies), 1% GlutaMAX (Invitrogen), 10 ng ml⁻¹ IL-3 (Peprotech), 10 ng ml⁻¹ IL-6 (Peprotech) and 25 ng ml⁻¹ SCF (Peprotech); the cells were maintained at a concentration of 0.1 × 10⁶ cells ml⁻¹. After 7 d, the cultures were divided for erythroid and myeloid differentiation. Erythroid differentiation of cells at a concentration of 0.2 × 10⁶ cells ml⁻¹ was induced using stem cell medium supplemented with 2 U ml⁻¹ erythropoietin (Peprotech). Myeloid differentiation of cells at 0.3 × 10⁶ cells ml⁻¹ was induced in Myelocult H5100 medium (StemCell Technologies) supplemented with 1 × 10⁻⁶ M hydrocortison (StemCell Technologies) and 20 ng ml⁻¹ G-CSF (Peprotech). Differentiation was evaluated in healthy control BM samples by May–Grünwald Giemsa staining and flow cytometry as a positive readout. For erythroid and myeloid assessment, the following antibodies were used: anti-CD36–PE (1:200; BioLegend), anti-CD235a–PECy7 (1:1,000; BioLegend), anti-CD33–APC (1:200; BioLegend), anti-CD66b–FITC (1:200; BD Biosciences) and anti-CD34–BV421 (1:100; BioLegend).

To differentiate transgenic U937 cells to monocyte/macrophage, 1 × 10⁶ cells/mL were incubated with complete RPMI supplemented with a final concentration of 10 nM phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, Gillingham, UK) in 24 well plates for 48 h. To control the metabolic and redox contribution of BCAT1, the media was supplemented with 20 mM Gabapentin (Sigma-Aldrich, Gillingham, UK) and/or 10 mM NAC respectively. Intracellular ROS ± PMA was measured by DCFDA staining as previously described. To examine cellular differentiation, U937 cells were incubated with anti-CD14 FITC (Biolegend), anti-CD68 PE (Biolegend), anti-CD36 PE (Biolegend, London, UK) and anti-CD11b FITC (Biolegend, London, UK) prior to flow cytometric analysis, where % positive cells and median fluorescence intensity (MFI) were evaluated. PMA treated cells were stained with Giemsa and imaged via light microscopy to evaluate macrophage morphology.