Fig. 5
Reduction of cysteine synthase activity induces xCT expression by up-regulating NRF2. A, The transporting activity of xCT in normal hepatocytes and BNL 1ME A.7 R.1 cells prepared from tumor-bearing mice. Each value represents the mean with S.D. (n = 4). **P < 0.01; significant difference compared with the normal hepatocytes at the corresponding times (F2,18 = 7.921; P < 0.001; Two-way ANOVA with Tukey–Kramer’s post hoc test). B, Intracellular cysteine contents in primary hepatocytes and BNL 1ME A.7 R.1 cells after treatment with 1.5 µM erastin. Each value represents the mean with S.D. (n = 6). **P < 0.01; significant difference between the two groups (F3,20 = 112.712, P < 0.001; ANOVA with Tukey–Kramer’s post hoc test). C, The mRNA levels of Slc7a11 in primary hepatocytes after treatment with 1.5 mM AOAA or 1.0 mM PAG for 24 h. Each value represents the mean with S.D. (n = 4). **P < 0.01; significant difference between the two groups (t6 = 7.297, P < 0.001; unpaired t-test, two sided). D, The protein levels of xCT in primary hepatocytes and BNL 1ME A.7 R.1 cells after treatment with 1.0 mM PAG for 24 h. Each value represents the mean with S.D. (n = 4). **P < 0.01; significant difference between the two groups (t4 = 7.301, P = 0.002; unpaired t-test, two sided). E, The protein levels of ATF4 and NRF2 in the liver and hepatic tumors. Each value represents the mean with S.D. (n = 4). *P < 0.05; significant difference between the two groups (t6 = 2.748, P = 0.033; unpaired t-test, two sided). F, The protein levels of xCT and NRF2 in mock-transfected or Nrf2-knockdown (KD) BNL 1ME A.7 R.1 cells. Each value represents the mean with S.D. (n = 4). **P < 0.01, *P < 0.05; significant difference between the two groups (t6 = − 7.171, P < 0.001 for xCT; t6 = − 3.664, P = 0.011 for NRF2; unpaired t-test, two sided). G, The protein levels of NRF2, xCT and phosphorylation states of AKT protein in BNL 1ME A.7 R.1 cells after treatment with 10 µM Akti X for 48 h. The protein levels were normalized to those of β-ACTIN (for NRF2 and xCT) or total AKT (for p(S473) AKT and p(T308) AKT). The values in vehicle-treated cells were set at 1.0. Each value represents the mean with S.D. (n = 4). **P < 0.01, *P < 0.05; significant difference between the two groups (t6 = − 5.694, P = 0.013 for p(S473) AKT; t6 = − 3.227, P = 0.018 for p(T308) AKT; t6 = − 3.874, P = 0.008 for NRF2; t6 = − 4.126, P = 0.006 for xCT; unpaired t-test, two sided)