Extended Data Fig. 10: CKB-mediated GPX4 phosphorylation promotes tumour growth.
From: Creatine kinase B suppresses ferroptosis by phosphorylating GPX4 through a moonlighting function
(a, b, e-i, k, l, n, o, q) Data are the mean ± SD, ^P < 0.05; *P < 0.01; **P < 0.001; ***P < 0.0001; N.S., not significant by two-tailed Student’s t-test (a, e, g, h, i, l, o) and by two-tailed Mann-Whitney U test (b, f, k, n, q). (b, f, j, m, p) IHC analyses of the indicated xenograft tumors from nude mice were performed with the indicated antibodies. Representative staining images and staining scores are shown. (a, b) Huh7 cells were subcutaneously injected into 6-week-old male athymic nude mice. When the tumor reached 50 mm3, the mice were assigned randomly into different treatment groups. Sulfasalazine (SAS) and Liproxstatin-1 (Lip-1) were intraperitoneally injected daily at a dose of 100 mg/kg and 10 mg/kg respectively until the endpoint at Day 28. Tumor volume and weight were analyzed (n = 7) (a). (c, r) The whole cell lysates of WT and GPX4 knockout Huh7 cells (c) and L02, THLE-2, Huh7, HCCLM3 (r) were harvested for immunoblotting analyses as indicated. (d-f) WT and GPX4 knockout Huh7 cells were subcutaneously injected into the left or right flanks of 6-week-old male athymic nude mice with and without daily Lip-1 intraperitoneal injection from the 4th day. The resulting tumors were resected 28 days after injection (d). Tumor volume and weight were analyzed (n = 6) (e). (g-q) IGF1R CA-expressing parental Huh7 cells and the indicated clones with knock-in expression of CKB T133A (g) and GPX4 S104A (h) or Huh7 cells expressing CKB shRNA with reconstituted expression of the indicated CKB proteins (i-k) or Parental Huh7 cells and the indicated clones with knock-in expression of GPX4 S104D mutants stably transfected with CKB shRNA and reconstituted with indicated CKB proteins (l-q) were intrahepatically (g, h) or subcutaneously (i-q) injected into 6-week-old male athymic nude mice (n = 7). When the tumor reached 50 mm3, the mice were assigned randomly into different treatment groups. SAS was intraperitoneally injected daily at a dose of 100 mg/kg until the endpoint at Day 28. Tumor volume and weight were analyzed.
