Extended Data Fig. 6. PCK2 directly phosphorylates ACSL4 and promotes its activity.
a, In vitro kinase assays were performed by mixing Escherichia coli-purified His-ACSL4 (AA552-711) and 293T-purified active Flag-PCK2 with or without λ-Phosphatase. b, Schematic of PCK2 full-length and truncated mutants. c, HEK293T cells were transfected with the indicated Myc-tagged constructs, the ACSL4-PCK2 interaction was analyzed by immunoprecipitation with anti-Myc beads and immunoblotting. d, Immunoblots showing the expression of PCK2, ACSL4 and ACSL4 pT679 in HONE1 cells that expressing PCK2 wild type (FL) or catalytic domain-deletion mutation (ΔMD). e, PCK2-knockout HONE1 cells and parental cells were treated with AA-d8 (10 µM) for 36 h. The relative changes of PC that contain AA (20:4)-d8 are shown. n = 4. * P < 0.05; ** P < 0.01; *** P < 0.001. P = 0.0157, 0.0004, 0.0047, 0.0002, 0.0395, 0.0009, 0.0124, 0.0103, 0.036, 0.0085, 0.0076 and 0.0048. f, Radar chart indicates the changes of SFA-PLs, MUFA-PLs, and PUFA-PLs in PCK2-knockout cells and parental cells (upper). The relative increment of these phospholipids was represented by a bar chart (lower). g, Principal component analysis (PCA) of oxygenated phospholipids in PCK2-knockout cells and parental cells in the present or in the absence of RSL3. h, LC-MS-based heatmap showing relative changes in RSL3-induced oxygenated PE molecular species in PCK2-knockout HONE1 cells and parental cells. Row z-scores were obtained from averaged values of the content of lipid species. i, Dose-dependent toxicity of FINO2 induced cell death of PCK2-knockout HONE1 cells and parental cells. j, Percentage of dead cells in PCK2-knockout HONE1 cells and parental cells treated with IR or Cis. Data are shown as mean ± SD (e, j), unpaired two-tailed t-test (e) or one-way ANOVA (j), n = 3 independent experiments. One of three experiments is shown (a, c-d).