Figure 6. Dipeptide synthesis protects KEAP1 mutant cells from ferroptosis.

(A) Correlation between cell death under cystine starvation (AUC, from Figure 1A) and GCLC expression in NSCLC cells (N=13). GCLC expression is normalized to HSP90 expression and can be found in Figure 5A. (B) Evaluation of the influence γ-Glu-dipeptide treatment on the death of GCLC KO A549 cells under cystine starvation. Cells were treated with γ-Glud-ipeptides at the indicated concentration or Fer-1 (10 μM) as a positive control. Cell death was monitored with Sytox Green every 2 hrs for 49 hrs, normalized to cell density, and then AUCs were calculated (N=3). (C) Evaluation of the death of NRF2^HIGH (N=7) and NRF2^LOW (N=10) cell lines under cysteine replete and starved conditions in the presence and absence of 100 μM BSO. Each dot represents the mean AUC of each NSCLC cell line. Cell death was monitored with Sytox Green and individual cell line data is found in Figure S6B. (D) Evaluation of the death of NRF2 KO A549 cells transduced with empty vector (EV) or NRF2 under cystine replete and starved conditions in the presence and absence of 100 μM BSO (N=3). Cell death was monitored by Sytox Green every 2 hours for 65 hours, followed by AUC calculation. (E) Evaluation of the death of Calu3 cells pre-treated with 100 nM of KI-696 or vehicle (Veh, 0.1% DMSO) for 48 hrs, followed by culture under cystine replete and starved conditions in the presence and absence of 100 μM BSO (N=3). Cell death was monitored with Sytox Green every 2 hours for 47 hours, followed by AUC calculation. (F) Evaluation of the death of parental A549 cells, GCLC KO clones reconstituted with GFP (+GFP) or sgRNA-resistant GCLC (+GCLC_Res), and GSS KO clones reconstituted with GFP (+GFP) or sgRNA-resistant GSS (+GSS_Res) following cystine starvation for the indicated time points (N=3). (G) Evaluation of the death of GCLC KO clones reconstituted with GFP (+GFP) or sgRNA-resistant GCLC (+GCLC_Res), and GSS KO clones reconstituted with GFP (+GFP) or sgRNA-resistant GSS (+GSS_Res) treated with of Erastin (5 μM) for the indicated time points (N=3). (H) Evaluation of the death of the cells from (G) under the cystine starved conditions in the presence and absence of 100 μM BSO. (N=3). (I) Evaluation of the death of H1299^Cas9 cells infected with sgRNAs (sgCon, sgGCLC or sgGSS), followed by reconstitution with GFP (+GFP), sgRNA-resistant GCLC (+GCLC_Res), or sgRNA-resistant GSS (+GSS_Res) under cystine starved conditions (N=4). For F-H, results are representative of 2 independent GCLC and GSS KO clones. For F-I, cell death was monitored by Cytotox Red (CR) every 2 hrs (I), 3 hrs (F, G) or 4 hrs (H). For B-I, data are presented as mean ± SD. N is number of biological replicates. *P<0.05, ***P<0.001, and ****P<0.0001. For A, a Pearson correlation analysis was used. For B-E, a one-way ANOVA with Bonferroni’s multiple comparison test was used for statistical analyses.