Lysine hcl

All cell culture reagents were obtained from Gibco unless otherwise stated. Cells were cultured for at least seven doublings in SILAC DMEM supplemented with 10% Dialyzed FBS, 20 Units/mL Penicilin, 20 µg/ml Streptomycin, 1 mM Sodium Pyruvate, 10%, and 2 mM L-alanyl-L-glutamine dipeptide and either; 42 mg/L ¹³C₆,¹⁵N₄-L-Arginine HCl (Silantes) together with 73 mg/L ¹³C₆,¹⁵N₂-L-Lysine HCl (Silantes), or 42 mg/L Arginine HCl and 73 mg/L Lysine HCl with standard isotopic constituents (Sigma). Cells were harvested by rinsing twice in ice-cold PBS excluding Calcium Chloride and Magnesium Chloride. Cells from 1 × 10 cm dish were scraped in 1 ml of ice-cold PBS and transferred to a 1.5 mL Eppendorf tube, for centrifugation at 300 x g at 4°C. The PBS was aspirated and cells were resuspended in lysis buffer (4% SDS, 100 mM DTT in 100 mM Tris-HCl, pH 7.6 at room temperature) and heated to 95°C for 5 min. Samples were sonicated for 15 × 30 s cycles using a Bioruptor to reduce viscosity of the lysate. Protein concentrations were determined via tryptophan assay.

Cells were seeded at the density of 400 cells/well. A day after seeding, cells were treated with rapamycin or sildenafil. Cells were refed with arginine depleted or arginine containing media 2 days after seeding. Clonogenic survival assays of arginine-depleted cells were conducted in SILAC media (Gibco, A2493901) supplemented with 5-mM glucose, 1-mM sodium pyruvate (Sigma Aldrich, S8636), 4-mM glutamine (Agilent, 103,579–100), phenol red (Sigma-Aldrich, P0290), and lysine-HCl (Sigma-Aldrich, 444,208) to emulate arginine-free DMEM (11,885). Medium for control groups of arginine depletion clonogenic assay was prepared by adding arginine-HCl (Sigma-Aldrich 08163). After about 10 days when visible colonies defined as cluster have formed, plates were scanned and colonies counted using ImageJ.