13c6 arginine

Cis-vaccenic acid (> 97 % purity) and Conjugated linOleic acid (> 96 % purity) were purchased from Cayman Chemicals. ¹³C₆Arginine and ¹³C₆¹⁵N₄Arginine (both 99 % purity) were from Cambridge isotope laboratories (Andover, MA, USA). Elaidic acid, Oleic acid, trans-vaccenic acid , L-Lysine and Arginine (all 99 % purity), Anhydrous dimethyl formamid (99.5 % purity), iodoacetamide, tetra-methyl-ethylene-diamine, L-Glutamine, Human serum albumin and RPMI-1640 without Arginine, Lysine and Leucine were purchased from Sigma. Regular SynQ serum substitute and SynQ without Arginine and Lysine was from Cell Culture Service, Hamburg Germany. All reagents for DIGE were PlusOne quality from GE Healthcare. Also CyDye DIGE fluors, IPG running buffers and PD10 columns were purchased from GE Healthcare. Penicillin, Streptomycin, regular RPMI-1640 and CyQuant NF proliferation assay kit were from Invitrogen.

Primary CML samples were thawed and recovered overnight using physiological medium (Plasmax) (Vande Voorde et al, 2019 (link)). This medium was supplemented with labelled or non‐labelled nutrients as well as standard supplements and growth factors as described previously (Kuntz et al, 2017 (link)), then filter sterilised through a 0.2 μM filter (Fisher Scientific: 10509821). Stable isotope tracers were purchased from Cambridge Isotopes (¹³C₆ Arginine: Cat# CLM‐2265, ¹³C₅ Ornithine: Cat# CLM‐4724 and ¹³C₅ Citrulline: Cat# CLM‐8653) and added at concentrations found in Plasmax. Primary samples were seeded at a density of 400,000 cells/ml and cell lines at 100,000 cells/ml. For cell lines, the medium was supplemented with 10% dialysed FBS (Thermo Fisher Scientific Cat# A33820‐01). Imatinib was purchased from LC Laboratories and BCT‐100 was supplied from BCT International. The CD34⁺ cells were isolated using the CliniMACS (Miltenyi Biotec) to 95% purity while normal CD34⁺ cells (> 90% purity) using human CD34 MicroBeads (Miltenyi Biotec: Cat# 130‐100‐453), according to manufacturer's instructions.

Male C57BL/6 LDLr^−/− mice were purchased from Jackson Labs, Bar Harbor, ME. Authentic and isotopically labeled standards were purchased from the following vendors: NMMA, SDMA and D₆ SDMA (Santa Cruz, CA); D₇ ADMA (Novachem, Australia); Creatinine, D₃ Creatinine, Arginine and Citrulline (Sigma-Aldrich); ¹³C₅-Citrulline and ¹³C₆-Arginine (Cambridge Isotope Laboratories, MA). All LC reagents were purchased from Sigma Aldrich, St. Louis, MO. Rodent diets were purchased from LabDiet^® and high fat diet from Harlan Teklad.

HeLa cells were split from normal growth media into arginine and lysine-free DMEM (Caisson Laboratories Inc.) supplemented with 10% heat inactivated dialyzed FBS (Gibco) and either 1 mM ²H₄-lysine and 0.1 mM ¹³C₆-arginine (Cambridge Isotope Laboratories) for heavy cells or normal isotopic abundance of l-lysine and l-arginine (Sigma) for light cells. Cells were maintained in labelling media for at least 5 cell divisions to ensure complete labelling as described elsewhere^{28 (link)}.

NOZ cells were grown in a ¹³C₆-lysine/¹³C₆-arginine-containing (heavy) medium, while NOZ GemR cells were grown in a normal (light) medium. The experiment was carried out in a biological duplicate. DMEM with and without lysine and arginine, fetal bovine serum (FBS), L-glutamine, and antibiotics were purchased from Thermo Fisher Scientific. SILAC amino acids, ¹³C₆-lysine, and ¹³C₆-arginine were acquired from Cambridge Isotope Laboratories (Andover, MA, USA). Peptides were prepared using an in-solution tryptic digestion protocol with modifications [54 (link),55 (link)]. The eluted peptides were lyophilized and subjected to phosphopeptide enrichment by immunoaffinity purification (IAP), as previously described [54 (link),55 (link)]. Peptides were eluted twice from beads by incubating the beads with 0.1% TFA at room temperature. Protocol details are shown in the Supplementary Materials: Supplementary Methods/SILAC protocol.