Extended Data Fig. 5: Hydroxyestrogens impair mitochondrial acetyl-CoA production and histone acetylation required for LPS-induced proinflammatory gene transcription.
From: Mitohormesis reprogrammes macrophage metabolism to enforce tolerance
a. BMDMs pretreated 1h with EtOH or 4-OHE1 before 6h LPS stimulation and qPCR. The average percent induction at each 4-OHE1 concentration relative to control for each gene was calculated (n = 3 independent biological replicates) and plotted. b. Abundance (area under the curve, AUC) of TCA cycle metabolites in EtOH versus 4-OHE1-treated BMDMs. c. Fractional contribution (FC) of 13C6-glucose-derived carbons to TCA cycle metabolites and amino acids in EtOH versus 4-OHE1-treated BMDMs (significant labeling in bold). d. UCSC genome browser H3K27ac ChIP-seq tracks displaying regions of decreased read density (arrows) in the Il1b, Cd69, Ifitm3, and Nfkbid loci. Read density was locally scaled, and black bar added to provide common reference point across tracks. e. RAW macrophages cultured in CoA (500 μM, top) or acetyl-CoA (Ac-CoA, 500 μM, bottom) (red bars) for 3h prior to 1h EtOH or 5 μM 4-OHE1 pretreatment, 6h LPS stimulation, and Il1b qPCR. f. RAW macrophages cultured in sodium acetate (5mM, purple bar) or CoA (500 μM, red bar) for 15 minutes prior to 1h EtOH or 5 μM 4-OHE1 pretreatment, 1h LPS stimulation, and Il1b qPCR. g. Data from reference24. Acetyl-CoA levels in HeLa cells treated with vehicle or 10 μM FCCP for 24h. n = 7 independent biological replicates per condition. h. RAW macrophages pretreated with vehicle or itaconate (7.5mM) for 1h before 3h LPS stimulation and Il1b qPCR. i. RAW macrophages cultured in acetyl-CoA (Ac-CoA, 200 μM, bottom) for 2h prior to pretreatment with vehicle or itaconate for 1h before 3h LPS stimulation and Il1b qPCR. Each data point is an independent biological replicate. For n = 2, data represented as mean; for n = 3 or more, data represented as mean ± SEM. All P values from unpaired, two-sided Student’s T Test (planned comparisons). qPCR data representative of at least 2 independent experiments. Metabolomics and ChIP-seq studies were performed once.
