L leucine

MEF and HT1080 cells were maintained in our laboratory. These cells were cultured in DMEM medium (8122292, Gibco, Billings, MT, USA) supplemented with 10% fetal bovine serum (SH30406, Hyclone, Logan, UT, USA) and 1% penicillin/streptomycin (SV30010, Hyclone), and placed in a humidified incubator with 5% CO₂ at 37 °C. In the case of arginine deprivation, cells were grown in DMEM/F-12 medium without L-arginine, L-leucine, and L-lysine (D9811-15D, US Biological, Salem, MA, USA) supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin as well as 59.05 mg/L L-leucine (61-90-5, Sigma, St. Louis, MO, USA) and 91.25 mg/L L-lysine (657-27-2, Sigma). In the case of the control group with the complete medium, 59.05 mg/L L-leucine, 91.25 mg/L L-lysine, and 147.5 mg/L L-arginine (1119-34-2, Sigma) were added to the DMEM/F-12 medium without L-arginine, L-leucine, and L-lysine.

Primary muscle progenitor cells (MPs) were isolated from muscle tissue as described in the Supporting Information. A total of 200 000 cells were seeded in six‐well plates. Myogenic differentiation medium containing low‐glucose Dulbecco's modified Eagle medium, 2% horse serum (Invitrogen), and 1% P/S was added 24 h later. Fully differentiated MPs were treated 72 h later according to one of the following conditions: (1) STV, 1 h Dulbecco's modified Eagle medium w/o amino acids (Biomol GmbH) + 10% dialyzed fetal bovine serum (Invitrogen) + 1% P/S; (2) LEU 0.8, 1 h STV + 1 h 0.8 mM L‐Leucine (Sigma‐Aldrich); and (3) LEU 5, 1 h STV + 1 h 5 mM L‐Leucine. Experiments were performed in biological triplicate and technical duplicate. 5‐Ethynyl‐2´‐deoxyuridine (EdU) analysis was performed according to manufacturer's protocol (Thermo Fischer Scientific) after a 4 h pulse with 1 μg/mL EdU. For proliferation analysis, 20 000 cells were plated on 12‐well plates, and three plates were counted per time point over 5 days.

For in vitro studies, cells from Walker-256 tumour-bearing animals were isolated from the intraperitoneal implant and maintained in culture. Briefly, the ascites fluid from a tumour intraperitoneal implant was collected, and the erythrocytes were lysed with 55 mM NH4Cl, 12 mM NaHCO3, and 0.1 mM EDTA, and the cell suspension was centrifuged at 500 × g, for 5 min, 4 °C. The supernatant was removed, and the pellet containing the tumour cells was seeded in 199 medium (Sigma-Aldrich) supplemented with 10% bovine calf serum (Lonza) and 1% penicillin/streptomycin (Lonza) and maintained at 37 °C and 95% O₂–5% CO₂ atmosphere with 85% relative humidity. Lactate production and glucose consumption in vitro analyses were done as a measurement of the lactate concentration released in the medium, and the glucose consumed was measured accordingly to the manufacturer’s instructions (Bioclin, Brazil). Briefly, cells were cultured in 12-well plates and treated with 50 µM L-leucine (Sigma, USA) for 24 h. The medium was removed and assayed for lactate production^{1 (link)} and glucose consumption^{2 (link)}. Additional Walker-256 cells were seeded in 12-well plate and treated with 50 µM L-leucine (Sigma, USA) for 24 h for mitochondrial respiratory function analyses (Seahorse), protein extraction (western blotting), and RNA extraction (qPCR).