Arginine and lysine

Cells were plated at 21,000 cells per cm² in 6-well plates in normal growth media. After 24 h cells were washed twice with phosphate buffered saline (PBS) and treated with the respective starvation or non-starvation media. For the initial screening, DMEM or SILAC RPMI 1640 medium (Gibco) lacking glucose and glutamine were supplemented with 10 mM or 0.2 mM glucose (Sigma-Aldrich, St. Louis, MO, USA), 2 mM glutamine and 0% or 10% dialyzed FBS (Gibco). SILAC RPMI was additionally supplemented with arginine and lysine (Sigma-Aldrich) at concentrations present in normal RPMI 1640. For serine/glycine and glucose starvation, DMEM without glucose, glutamine, serine and glycine, containing standard cystine (Biomol, Hamburg, Germany) was supplemented with serine, glycine and glucose (Sigma-Aldrich) at the indicated concentrations. glutamine was added at a concentration of 2 mM. In some experiments, custom-made DMEM medium lacking glucose, glutamine, serine, glycine, cystine, cysteine, pyruvate and phenol red (Cell Culture Technologies, Gravesano, Switzerland) was used. For the supplementation with different levels of cystine, a 100 mM stock in 1 M HCl was freshly prepared. During the course of experiments media were refreshed every 24 h, unless indicated otherwise.

HeLa (ATCC: CCL-2) cells were tested for mycoplasma contamination and grown in DMEM supplemented with 10% FBS, 2 mM L-glutamine, and 1% penicillin/streptomycin. SILAC media was supplemented with arginine and lysine (SILAC Light) or with heavy isotope-labeled arginine (¹³C_6,¹⁵N₄-arginine, Sigma) and lysine (¹³C_6,¹⁵N₂-lysine, Cambridge Isotope Laboratories) (SILAC heavy) in media containing dialyzed serum (Sigma). Cells were cultured at 37 °C in a humidified incubator at 5% CO₂. At a confluency of ~90%, cells were washed twice with PBS and lysed in ice-cold modified RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1x mini complete protease inhibitor cocktail (Roche), 10 mM nicotinamide, and 5 μM trichostatin A). Lysates were mixed with 1/10 volume of 5 M NaCl to release chromatin-bound proteins and incubated for 15 min on ice. Subsequently, lysates were homogenized by sonication (6 × 10 sec, 15 W), cleared by centrifugation (20,000 × g, 15 min, 4 °C), and the supernatant precipitated by addition of four volumes of −20 °C acetone. Precipitates were re-dissolved in 8 M guanidine HCl, 50 mM Hepes pH8.5 and protein concentration was determined by Quick-start Bradford assay (Bio-Rad).