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. 2022 Jun 8;606(7916):968–975. doi: 10.1038/s41586-022-04787-x

Extended Data Fig. 1. (related to Figure 1): (a-e) FAHFA biosynthesis is sensitive to fluorophosphonate inhibitors; (f) ATGL protein is increased in AG4OX mice adipose tissue.

Extended Data Fig. 1

(a) 9-PAHSA biosynthesis in 3T3-L1 adipocytes pre-incubated with the indicated concentrations of methyl arachidonyl fluorophosphonate (MAFP) or 0.1% DMSO (Veh) for 1hr, and then co-incubated with C17:1 and 9-HSA or 0.1% DMSO (Veh) for 2hrs. (n = 4 wells, except for MAFP 6.75 µM group n = 3, mean±SEM), *P < 0.0001 vs. C17:1 and 9-HSA only group (One-way ANOVA) (b) 9-POHSA biosynthesis in WT and AG4OX SVF-adipocytes pre-incubated with MAFP or 0.1% DMSO for 1hr, then co-incubated with 9-HSA or 0.1% DMSO (Veh) for 2hrs. (n = 6 wells, mean±SEM) *P < 0.05, **P < 0.0001 vs. WT, same treatment, t-test correcting for Holm-Sidak multiple comparisons. #P < 0.001, ##P < 0.0001 vs. the Veh for 9-HSA, same genotype, @P < 0.05 vs. 9-HSA alone, same genotype (Two-way ANOVA). (c) 9-PAHSA and (d) 9-POHSA biosynthesis in WT and AG4OX SVF-adipocytes pre-incubated with FP-alkyne (5 µM) or 0.1% DMSO for 1hr, then co-incubated with 9-HSA (10 µM) for 2hrs. (n = 4 except for AG4OX Vehicle group = 6, mean±SEM) *P < 0.001 vs. WT, same treatment, #P < 0.01 vs. Veh, same genotype (Two-way ANOVA). (e) Selected list of candidate FAHFA biosynthetic enzymes from activity-based proteomics study in WT and AG4OX SVF adipocytes. (f) WT and AG4OX SQ WAT western blots for ATGL and GAPDH. Total protein between 37 and 25 kDa was visualized by Revert stain (N = 8/genotype). Loading control, GAPDH, and ATGL were run on the same blot. This blot is representative of 3 separate blots. ND means not detected.