Reagents were obtained from the following sources: Cell culture grade amino acids from Sigma; z-VAD-FMK, Necrostatin-1, Necrosulfonamide from Calbiochem; Necrox-5 from Santa Cruz Biotechnology Inc; Antibodies to phospho-p38, total p38, β-tubulin, claudin-1 and Vimentin (D21H3) from Cell Signaling Technology; RACK1 antibody from BD; Antibodies to TNFα and MEKK4 from Abcam; Noxa antibody from Novus Biologicals.
All breast tumor cells were cultured in DMEM with 10% heat-inactivated FBS, 1% penicillin-streptomycin in a humidified incubator at 37°C and 5% CO2. To prepare media deficient in each amino acid, Earle’s balanced salt solution was supplemented with 4.5 g/L glucose, 0.37 mM sodium bicarbonate, 24.8 μM ferric nitrates, 10% dialyzed FBS (Sigma) and MEM vitamin solution (Invitrogen). Different amino acid combinations were then added. Cells were plated in the complete DMEM media 1 day prior to PBS-rinsing and amino acid-deprivation.
All breast tumor cells were cultured in DMEM with 10% heat-inactivated FBS, 1% penicillin-streptomycin in a humidified incubator at 37°C and 5% CO2. To prepare media deficient in each amino acid, Earle’s balanced salt solution was supplemented with 4.5 g/L glucose, 0.37 mM sodium bicarbonate, 24.8 μM ferric nitrates, 10% dialyzed FBS (Sigma) and MEM vitamin solution (Invitrogen). Different amino acid combinations were then added. Cells were plated in the complete DMEM media 1 day prior to PBS-rinsing and amino acid-deprivation.