Extended Data Fig. 6: Hydroxyestrogen-driven mitochondrial stress triggers mitohormesis.
From: Mitohormesis reprogrammes macrophage metabolism to enforce tolerance
a. Oxygen consumption in RAW macrophages after treatment with vehicle or 4-OHE1. 5 repeated measurements per condition were taken over 45min and averaged (n = 4 independent biological replicates). b. Mitochondrial membrane potential in RAW macrophages treated 20min with vehicle, oligomycin, FCCP, or 4-OHE1 (5 μM). c. RAW matrix-oxGFP macrophages treated 8h with vehicle or 4-OHE1 and fluorescence quantified by flow cytometry. d. qPCR for matrix-oxGFP transcript in parental macrophages and RAW matrix-oxGFP macrophages treated 8h with vehicle or estrogens (5 μM). e. Mitochondrial DNA content in RAW macrophages treated with vehicle or 5 μM 4-OHE1. f. RAW matrix-oxGFP macrophages pretreated 1h with NAC (5mM) or MitoQ (1 μM) prior to 8h vehicle or 5 μM 4-OHE1 treatment. g. RAW matrix-oxGFP macrophages pretreated 1h with KRIBB11 (10, 7.5, 5 μM) before 8h treatment with 5 μM 4-OHE1. h. i. RAW macrophages and BMDMs treated 8h with vehicle or estrogens (5 μM) before MitoTracker Green staining and flow cytometry. j. THP-1 and U937 matrix-oxGFP cells treated 24h with vehicle or estrogens (5 μM). k. BMDMs pretreated 1h with vehicle or estrogens (5 μM) before 6h LPS stimulation, MitoPY1 staining, and flow cytometry. l. left – roGFP experimental strategy. right – RAW matrix-roGFP 405nm/488nM excitation ratio after H2O2 and DTT treatment. m. RAW macrophages pretreated 1h with NAC (5mM) or MitoQ (1 μM) prior to vehicle or 5 μM 4-OHE1 treatment (18h). Cells were then treated 2.5h with vehicle or 50 μM menadione before viability assessment by flow cytometry. n. BMDMs treated overnight with vehicle or 5 μM 4-OHE1 before vehicle (4h) or 50 μM menadione (2,4h) treatment and viability assessment. o. THP-1 and U937s treated 7h with vehicle or estrogens (5 μM) before H2O2 challenge, CM-H2DCFDA staining, and flow cytometry. 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). All data representative of at least 2 independent experiments.
