L arginine and l lysine

Hela and human skeletal muscle (hsSKM) cells were purchased from ATCC. HEK293A cells were purchased from Thermo Fisher. These cells were grown in DMEM (Biological industries, Israel) supplemented with 10% FBS (Biological industries, Israel) and 1% penicillin-streptomycin. Cells were maintained at 37°C and 5% CO₂. Cells were transfected with polyethylenimine (PEI) or LipoGene 2000 Star Transfection Reagent (US Everbright). Mouse embryonic fibroblast (MEF, ATCC) cells were grown for at least six generations in Dulbecco’s modified Eagle’s medium (DMEM) for SILAC (Thermo Fisher) supplemented with L-lysine and L-arginine (light) or L-lysine-13C6 and L-arginine-13C6, 15N4 (heavy) (Thermo Fisher) as previously described (Wang J. and Nakamura F. 2019 (link)). 50 mg of each amino acids was added into every 500 mL DMEM for SILAC.

A549, H82, H1650, and DM122 cells were maintained in RPMI 1640 (Fisher Scientific) and HEK293T cells were maintained in DMEM (Fisher Scientific) supplemented with 10% FBS (Omega Scientific, US Source Fetal Bovine Serum), 2 mM l-glutamine (Thermo Fisher Scientific), 10 units and 100 μg mL^–1 penicillin–streptomycin (Sigma-Aldrich). SILAC A549 and HEK293T cells were maintained in RPMI 1640 and DMEM media, respectively, for SILAC without l-lysine and l-arginine (Thermo Scientific) supplemented with 10% dialyzed FBS (Omega Scientific, US Source Fetal Bovine Serum), 2 mM l-glutamine (Thermo Fisher Scientific), 10 units and 100 μg mL^–1 penicillin–streptomycin (Sigma-Aldrich). ‘Light SILAC’ media was supplemented with l-lysine and l-arginine (100 μg mL^–1, Acros Organics). ‘Heavy SILAC’ media was supplemented with isotopically labeled l-lysine (¹³C₆, ¹⁵N₂) and l-arginine (¹³C₆, ¹⁵N₄, 100 μg mL^–1, Sigma-Aldrich) for a minimum of five passages prior to use. Cells were cultured at 37 °C in 5% CO₂.

HEK 293T cells obtained from CCTCC (China Center for Type Culture Collection, Wuhan, China) were grown in Dulbecco’s modified Eagle’s medium (DMEM, GIBCO, Grand Island, NE, USA) supplemented with 1% penicillin/streptomycin (GIBCO) and 10% fetal bovine serum (FBS, GIBCO) at 37 °C with 5% CO₂. The HSV-1 strain 129 (H129) was used in this study, and virus was stocked at −80 °C.
For SILAC labeling, HEK 293T cells were cultured in a SILAC medium (Thermo, Waltham, MA, USA) containing 10% dialyzed FBS (Thermo) and l-arginine and l-lysine (R0K0, “light”), l-arginine-13C6 and l-lysine-4,4,5,5-d4 (L) (R6K4, “medium”), or l-arginine-13C6, 15N4 and l-lysine-13C6, 15N2 (R10K8, “heavy”) (Sigma, St. Louis, MO, USA). Cells were cultured in the SILAC medium for at least seven doublings to ensure that the labeled amino acids were incorporated into cellular proteins. Cells cultured in different mediums were infected with HSV-1 at an MOI of 5 or mock treated, and harvested at 4 or 20 h p.i. Then, mock-treated cells (light), HSV-1 infected cells harvested at 4 h p.i. (heavy), and HSV-1 infected cells harvested at 20 h p.i. (medium) were mixed in an equal amount.