Figure 6. Glutamine Restriction and xCT Inhibition Increase ROS.

Icon codes in figure keys; NAC, N-acetylcystine; SASP, Sulfasalazine. Icons represent mean values+/−SD. Amino acid quantitation by HPLC. (A) Change in media cystine (xaxis) and glutamate (y-axis) concentrations of cells cultured 24 hours in control media. (B) 24 hrs of glutamine restriction or SASP treatment reduces cystine / glutamate exchange by exemplar Group C auxotrophs; cys, cystine; glu, glutamate. (C) Effects of 24 hour glutamine restriction or SASP treatment on GSH content in an exemplar Group C TNBC; Q-, glutamine-free media; SASP, SASP treatment in complete media; SASP + 2me, SASP treatment in the presence of beta mercaptoethanol. (D) ROS levels in basal carcinomas assessed by DCFHDA fluorescence, normalized to control media reactivity. Light blue, 2 day cultures in glutamine-free media; Group averages; A, 112% +/− 21; B, 121% +/−31; C,162% +/−59; t-test group A vs. C p=0.07; Group A vs. B+C p= 0.074. Light gray, glutamine-free media +NAC. Teal, cultures treated 24 hours with SASP; Group averages; A, 205% +/− 43; B, 287% +/− 62; C, 405% +/− 168; t-test Group A vs. C p= 0.019; Group A vs. B+C p= 0.003. Dark gray, SASP +NAC. (E) Heatmap of genes involved in xCT function; red, increased; green, decreased; N, purified normal CD10⁺ and BerEp4⁺ breast epithelial cells; I, proliferating non-tumorigenic cell lines; PE, ER⁺ tumor cells purified from pleural effusions. (F) SLC7A11 GeneChip hybridization signal (log₂) vs. cystine consumption, icons coded by molecular subtype. (G) ROS levels in M231, 48 hours after targeting SLC7A11 or a scrambled siRNA, in the presence or absence of N-acetylcystine (NAC). (H) SLC7A11 knockdown efficiency of siRNAs used in part G. See also Figure S6.