Extended Data Fig. 8: Mitohormesis in macrophages involves metabolic reprogramming that enforces an LPS-tolerant state.

a. Basal and maximal OCR from RAW macrophage mitochondrial stress test Fig. 8c. Data is average of 3 repeated measurements prior to oligomycin injection, or following FCCP injection, respectively. b. Mitochondrial stress test in BMDMs treated 20h with EtOH, 4-OHE1 (5 μM), LPS, or both. n = 5 independent biological replicates per condition. c. Il1b qPCR in BMDMs treated overnight (18–24h) with EtOH, 4-OHE1 (5 μM), LPS, or both, before treatment wash out, recovery (1–2h), and 6h secondary LPS stimulation. Data is plotted as secondary LPS fold-induction versus cells with the same primary treatment but no secondary LPS. d. RAW macrophages treated overnight with EtOH, 4-OHE1 (5 μM), LPS, or both, before wash out, recovery, and 6h secondary LPS stimulation for MitoPY1 staining and flow cytometry. Data plotted as percent change in MitoPY1 fluorescence versus no secondary LPS control for each primary treatment. P values are from comparison of treatment vs. naïve control. e. BMDMs treated overnight with EtOH or estrogens (5 μM) before wash out, recovery, and 6h secondary LPS stimulation (100ng/mL, 2.5 μM nigericin added the last hour) for IL-1β ELISA. f. IL1B qPCR in THP-1s treated overnight with EtOH, LPS, or 4-OHE1 (5 μM) before wash out, recovery, and 6h secondary LPS stimulation. Data plotted as LPS fold-induction for cells with the same primary treatment but no secondary LPS. g. Mitochondrial membrane potential (mtMP) in RAW macrophages treated with LPS and stained with JC-9 for flow cytometry. Except for b, each data point is an independent biological replicate. For n = 2, data represented as mean; for n = 3 or greater, mean ± SEM. P values are from unpaired, two-sided Student’s T Test (planned comparisons). BMDM Seahorse and ELISA were performed once. All other data representative of 2 independent experiments.