Figure 3.

Growth in methionine is linked to induction of oxidative stress. (A) Serial dilution analysis of cells in the presence of different nitrogen sources and antioxidants. Wildtype, gcn2Δ, and gcn2Δ:GCN2 serial dilutions were spotted onto agar plates containing YNB, 2% dextrose, 10 mM of either ammonium sulfate or methionine, and one of the following antioxidants: 10 mM ascorbic acid, 10 mM N-acetylcysteine, or 200 μM of EUK-134. Plates were incubated at 30° for 2 days before imaging. Image shown are representative of three biological replicates. (B) Flow cytometry analysis of cells incubated in ammonium or methionine media supplemented with 2 mM hydrogen peroxide or 10 mM ascorbic acid, respectively. CellRox Deep Red Dye was used to detect intracellular ROS accumulation after 0 or 60 min of incubation. Data presented are the averages of two biological replicates.