Fig. 4

Nrf2 is a negative regulator of STING during metabolic reprogramming. a Lactate and glucose were measured in supernatants from PMDC05 cells stimulated with LPS (1 μg mL⁻¹) or gardiquimod (4 µg ml⁻¹) for 24 h. Data are the means and s.e.m. from one of two independent experiments performed in septuplicates. b, c Itaconate was measured in the cell medium and in the cell pellets of LPS-stimulated PMDC05 by LC/MS. Data are the means and s.e.m. from one of two independent. d PMDC05 stimulated with LPS (200 ng mL⁻¹) or gardiquimod (400 ng ml⁻¹) for 24 h were lysed and analysed for HIF1α, IFIT1, and Vinculin (VCL) as loading control. e PMDC05 cells were stimulated with gardiquimod (4 μg mL⁻¹), LPS (1 μg mL⁻¹), CpG (4 μg mL⁻¹), cGAMP (4 μg mL⁻¹), or Poly (I:C) (4 μg mL⁻¹) for 24, 48, or 72 h. Whole-cell extracts were analysed by immunoblotting. f TMEM173 mRNA was assessed by qPCR in PMDC05 stimulated with indicated agonists for 48 h. Data are the means ± s.e.m. of one experiment performed in triplicate. g PMDC05 cells were pre-treated with the Nrf2 inhibitor ML385 (20 µM) before stimulation with LPS (1 μg mL⁻¹) or gardiquimod (4 μg mL⁻¹) for 72 h. Cells were then lysed and lysates were analysed by western blotting. h PMDC05 cells were pre-stimulated with gardiquimod (4 μg mL⁻¹) or LPS (1 μg mL⁻¹) for 72 h before challenge with cGAMP (4 μg mL⁻¹) for 24 h. Type I IFN release was assessed using a HEK-Blue assay. Data are the means ± s.e.m. of one representative experiment performed in triplicate. i Graphical display of how 4-OI might possibly activate Nrf2. j HEK293T cells were treated with increasing doses of 4-OI (30–250 μM) for 18 h. ARE-promoter activity was assessed by luciferase assay. Data are means ± s.e.m. of two independent experiments in triplicate. k THP1 cells were treated with SFN (20 μM) or 4-OI (125 μM) for 48 h and mRNA levels were determined by qPCR. l THP1 cells were pre-treated with increasing does of 4-OI (62.5–125 μM) for 72 h and challenged with cGAMP (4 μg mL⁻¹). Whole-cell lysates were then blotted as indicated. Data are from one representative experiment that has been repeated twice. m–n THP1 cells were pre-treated with 4-OI (125 μM) for 72 h before challenge of 24 h with cGAMP (4 μg mL⁻¹). Supernatants were assessed for type I IFN by HEK-Blue cell assay (m) and whole-cell lysates by immunoblotting (n). o, p HaCat and A549 cells were treated with Nrf2 siRNA for 72 h and for 48 h, respectively, before treatment with 4-OI (125 μM) for 48 h. Whole-cell lysates were used for immunoblotting. q–s mRNA levels (q, r) and protein levels (s) were assessed by qPCR and immunoblotting in hMDMs silenced for Nrf2 by siRNA. Data are from two donors. Unpaired two-tailed Student’s t-test was used to determine significance