Figure 5. Inhibition of xCT Depletes Cysteine and Induces HIF.

(A) Immunoblot of Hs578T cells grown in fresh media supplemented with the indicated concentrations of L-cystine (L-Cys) and increasing concentrations of either L-glutamate (L-Glu; 300 and 800 µM), the xCT inhibitors (S)4-carboxyphenylglycine (4-CPG; 0.375 and 1 mM) or sulfasalazine (SAS; 94 and 250 µM), or the EglN inhibitor, DMOG (0.1 mM) for 4 hours.

(B) Immunoblot analysis of cells prepared as in Figure 4E.

(C) Representative dorsal bioluminescent images (BLI) of HIF1α-luciferase mice after two doses of 250 mg/kg sulfasalazine (SAS) or vehicle. Images were obtained 18 hours after the first dose.

(D) Cystine uptake in Hs578T cells grown in fresh media or media supplemented with 0.6 mM L-glutamate. Data are represented as mean ± SEM.

(E) Intracellular levels of the indicated metabolites, as determined by LC-MS, for MCF7 and Hs578T cells cultured in the presence of the indicated concentrations of L-cystine and L-glutamate. Data are represented as mean ± SEM, * refers to p-value < 0.05, and ** refers to p-value < 0.01.

(F) Immunoblot of Hs578T grown in media containing the indicated concentrations of L-cystine (L-Cys) and L-glutamate (L-Glu).

(G and H) Immunoblot analysis of Hs578T cells grown for 4 hours in media containing additives at the indicated concentrations, and for (G), increasing concentrations of L-cystine (L-Cys; to a final concentration of 0.4, 0.6 and 0.8 mM), N-acetylcysteine (NAC; 0.2, 0.5, and 1.0 mM) and β-mercaptoethanol (BME; 20, 50 and 100 µM).

(I) Change in FLuc activity, as determined by BLI, of orthotopic Hs578T cell tumors expressing FLuc driven by the 3xHRE promoter after treating mice with NAC, BHA, or their respective vehicles. For each mouse the values were first normalized to the BLI signal from a contralateral orthotopic Hs578T cell tumor expressing FLuc driven by a constitutive promoter (CMV) and then divided by the corresponding pretreatment ratio for that mouse.

See also Figures S4 and S5.