Figure 7. GLS1 Inhibitors Induce MYC-dependent Apoptosis.

(A) MYC activity was induced in HA1E-MYCER and IMR90-ERMYC cells as described in Fig. 6A. Cells then were treated with indicated concentrations of BPTES or 20767 or with 5 mM DON. 0.1% DMSO was used as a vehicle control for the experiments with BPTES. HA1E-MYCER cells were treated for 36 hours and IMR90-ERMYC cells were treated for 48 hours and percentage of apoptotic nuclei was scored. The average of three independent experiments is presented ± s.d. (B) HA1E cells transduced with either empty vector or vectors carrying either MYC or H-RAS^V12 were either incubated in glutamine-free media or treated with indicated concentrations of BPTES for 24 hours. Apoptosis was scored in three independent replicate samples. The values are given as the mean ± s.d. A representative of two independent experiments is shown. (C) HA1E-derived cell lines were infected with lentiviruses expressing either a scrambled shRNA (shScr) or two independent shRNAs targeting GLS1 (shGLS1_1 and shGLS1_2). Cells were collected 36 or 48 hours after the infection, GLS1 expression was evaluated by immunoblotting, and apoptosis was scored by counting apoptotic nuclei. For each shRNA the experiment was repeated at least twice. Transduction with empty pLKO.1 vector (EV) was used to account for the exceptional level of cell death induced by lentiviral infection observed specifically in HA1E cells with ectopic expression of MYC. (D) The ectopic expression of MYC and H-RAS^V12, as well as expression of ERK, P-ERK and GLS1 was estimated by western blotting in whole cell lysates. Increased expression of PERK indicates increased activity of RAS. (E) Indicated Burkitt’s lymphoma cell lines were cultured in the presence of 150 μM 20767. Cell death was analyzed by Annexin V and PI staining. A representative of two independent experiments is shown.