Extended Data Fig. 4: GPX4 S104 phosphorylation suppresses Lipid peroxidation.
From: Creatine kinase B suppresses ferroptosis by phosphorylating GPX4 through a moonlighting function
(a-d) Parental Huh7 and HCCLM3 cells and the indicated clones with knock-in expression of CKB T133A mutants (upper) or GPX4 S104A (lower) were treated with or without cystine deprivation (a, c) or 20 μM Erastin (b, d) and IGF1 for 24 h. Reduced GSH (a, b) and GSSG (c, d) were measured respectively. Data are the mean ± SD (n = 6), ^P < 0.05; *P < 0.01; **P < 0.001; ***P < 0.0001 by two-tailed Student’s t-test. (e) Parental Huh7 and HCCLM3 cells and the indicated clones with knock-in expression of CKB T133A mutants (left) or GPX4 S104A (right) were treated with or without cystine deprivation or 20 μM Erastin in the presence or absence of IGF1 (100 ng/ml) for 24 h. The cell lysates were harvested for immunoblotting analyses as indicated. (f-k) Parental HCCLM3 (f, g, j,k) or Huh7 (h, i) cells and the indicated clones with knock-in expression of CKB T133A or GPX4 S104A mutants were treated with or without cystine deprivation (f, h, j) or 20 μM Erastin (g, i, k) combined with Fer-1 (2 μM) in the absence or presence of IGF1 for 24 h. Lipid ROS-positive cells were measured. Data are the mean ± SD (n = 3), *P < 0.01 by two-tailed Student’s t-test (f, g). Lipid peroxidation was assessed using BODIPY 581/591 C11 staining followed by FACS analysis.
