GLS1 inhibition induces mitochondrial ROS formation and reduces GSH levels in DLBCL cells. (A) DOHH2 cells were treated with solvent or 500 nM CB-839 for 48 hours. Metabolites were analyzed and quantified by mass spectrometry and normalized to the solvent control. (B) DLBCL cells were treated with solvent or 500 nM CB-839 alone or in combination with 0.5 mM of membrane-permeable α-ketoglutarate (α-KG) for 5 days. Cell numbers were determined as indicated and normalized to the solvent control. (C) The indicated DLBCL cell lines were treated with the GSH-depleting agent DMF (20 μM) or 500 nM CB-839 for 24 hours alone or in combination with 0.5 mM membrane-permeable α-KG. The ratio of reduced to oxidized glutathione was determined and normalized to the respective solvent-treated controls. (D-E) DLBCL cells were treated for 48 hours with solvent or 500 nM CB-839 alone (D) or in combination with 100 μM α-tocopherol (E) before analysis of superoxide formation by flow cytometry using MitoSOX. The percentage of MitoSOX-positive cells in CB-839-treated samples was normalized to the solvent control. (F) WSU-DLCL-2 and U2932 cells were treated with solvent or 500 nM CB-839, alone or in combination with 100 μM α-tocopherol for 5 days. Cell numbers were determined as indicated and normalized to the solvent control. Error bars correspond to the mean ± SD. Data are representative of 3 independent experiments for panels A-F. (B-F) Statistical significance was calculated using the Student t test (unpaired, 2-tailed) to compare the indicated samples. DMF, dimethyl fumarate.