Fig. 4. Mitochondrial adenylate kinase 2 phosphorylates LOXL3 at S704 and confers resistance to chemo-induced ferroptosis.

a The workflow of the kinase screen for LOXL3-S704 phosphorylation which occurred in mitochondria. b The gray value was collected from the dot blot using an antibody specifically against pLOXL3-S704. The ratio of gray value by siNC versus siMK and analyzed as volcano. The red point indicates AK2. MK, mitochondrial kinase. c WB of Huh7 or Hep3B cells transfected with siRNAs targeting human AK2 using the indicated antibodies. d WB of Huh7 transfected with siRNAs targeting human AK2 and stimulated by EGF after starvation by removing serum for 12 h. e Huh7 or Hep3B cells expressing LOXL3-FLAG were collected for Co-IP and WB. f Lipid peroxidation determination of Huh7 or Hep3B cells transfected with siRNAs targeting human AK2 after treatment of Oxa-L for 12 h. g Huh7 cells transfected with siRNA targeting human AK2 were restored expression of gradually increasing mouse AK2 for 48 h and collected for WB. h LOXL3-FLAG was enriched and purified from Huh7 cells and then treated with λPP (1 μM) for half an hour to diminish S704-phosphorylation, then incubated with His-AK2 (WT or ED) in kinase reaction buffer. ED, enzyme dead, L199A/Q214A. i, j Huh7 or Hep3B cells transfected with siRNAs targeting human AK2 were restored expression with mouse WT or K14G/R17G mutant AK2 for 48 h and collected for WB confirmation(i). Meanwhile, respective lipid peroxidation determination was also performed (j). k Huh7 or Hep3B cells transfected with siRNA targeting human AK2 were restored expression of WT- or S704A-LOXL3 and treated with Oxa-L for the indicated time points and then cell viability was assessed. For (f, j, k), data present means ± SEM from three independent experiments (n = 3) and the statistical analysis was calculated by one-way ANOVA with Tukey’s HSD post hoc test (f, j) or two-way ANOVA for multiple comparisons (k). Source data are provided as a Source Data file. See also Supplementary Fig. 4.