A5006

U251 GB cells were seeded in wells of 96-well plates (5 × 10³ cells/well) overnight. Cells were co-incubated with Arginine (10 µM, A5006, Sigma) or an arginase inhibitor (nor-NOHA, 10 µM, 1140844-63-8, Cayman Chemical) for 24 h. The MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) cell viability assay was performed according to the manufacturer’s instructions (ab197010, Abcam), and cell viability was calculated using Prism 7.0 software (Prism GraphPad Software) according to the following formula: cell viability rate (%) = (OD490 of treated cells/OD490 of control) × 100%.

U2OS and U2OS Flp-In T-Rex cells were grown as adherent cells in Dulbecco's modified eagle medium (DMEM) depleted of arginine and lysine (Life Technologies, Carlsbad, CA A14431–01) supplemented with 10% dialyzed fetal bovine serum (Invitrogen, Carlsbad, CA 26400–044), 100 U/ml penicillin/streptomycin and 2 mm GlutaMax. Arginine and lysine were added in either light (Arg0, Sigma, A5006; Sigma-Aldrich (St-Louis, MO) Lys0, Sigma, L5501), medium (Arg6, Cambridge Isotope Laboratories, Inc. (Tewksbury, MA) (CIL), CNM-2265; Lys4, CIL, DLM-2640), or heavy (Arg10, CIL, CNLM-539; Lys8, CIL, CNLM-291) form to a final concentration of 28 μg/ml for arginine and 49 μg/ml for lysine. l-proline was added to a final concentration of 10 μg/ml to prevent arginine to proline conversion (see supplemental Fig. 1). Proteins were tested for >99% incorporation of the label after six passages by mass spectrometry (data not shown). U2OS stable cell lines were generated by transfecting pgLAP1 plasmids containing the cDNA of interest along with pOG44, the plasmid expressing the Flp-recombinase. Cells were then cultured with the addition of 150 μg/ml Hygromycin B and 15 μg/ml Blasticidine-HCl. For induction of DNA damage, the topoisomerase II inhibitor etoposide (#E1383, Sigma-Aldrich) was used at the indicated concentrations for 1 h, followed by wash with PBS and fresh normal media added.

Cells were homogenised in chilled 80% (v/v) methanol. Cell lysate samples were centrifuged at 12,000 rpm for 15 min and then transferred to a high recovery glass sample vial for vacuum drying at room temperature. The residue was oximated with 30 μl of pyridine containing 20 mg/ml methoxyamine hydrochloride (Sigma-Aldrich, 226904) at 37 °C overnight and further derivatised with 20 μl of N-tert-butyl dimethylsilyl-N-methyltrifluoroacetamide (Sigma-Aldrich, 394882) at 70 °C for 30 min. The derivatised sample was injected into an Agilent 1290 Infinity LC pump and a 6495 triple quadruple mass spectrometer (Agilent). Standard curves of commercial L-arginine (Sigma-Aldrich, A5006), L-ornithine (Sigma-Aldrich, O2375) or L-proline (Sigma-Aldrich, P0380) were used for quantification in the samples.

U2OS and U2OS Flp-In T-Rex (U2OS FT) cells were grown as adherent cells in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin/streptomycin and 2 mM GlutaMax. Additionally, U2OS Flp-In cells are maintained with 100 μg/ml Zeocin and 15 μg/ml Blasticidine-HCl. For culture in SILAC media, DMEM without arginine and lysine (Life Technologies A14431-01) was supplemented with 10% dialyzed fetal bovine serum (Invitrogen, 26400-044), 100 U/ml Penicillin/streptomycin and 2 mM GlutaMax. Isotopic arginine and lysine were added in either light (Arg0, Sigma, A5006; Lys0, Sigma, L5501) or heavy (Arg10, CIL, CNLM-539; Lys8, CIL, CNLM- 291) to a 28 μg/ml and 49 μg/ml final concentrations of arginine and lysine, respectively. To avoid proline to arginine conversion, an excess concentration of 10 μg/ml of L-proline was added to each medium.