Fig. 2: F3 is a type I IFN-stimulated gene with JAK-STAT-dependency, and DMF and 4-OI suppress TF release from macrophages via inhibition of type I IFN- and caspase-11-mediated pyroptosis.

a BMDMs were pre-treated with DMSO, DMF, or 4-OI (3 h) before LPS priming (16 h) and harvesting cell lysates (n = 6). Ifnb1 mRNA was quantified by qRT-PCR. DMSO–250 μM 4-OI, P = 0.00004. b BMDMs were pre-treated with DMSO, DMF, or 4-OI (3 h) prior to LPS priming (6 h). IFN-β in the supernatant was measured by enzyme-linked immunosorbent assay (ELISA) (n = 3). c BMDMs from WT and Ifnar^−/− mice (n = 4) were stimulated with LPS (3 h). F3 mRNA was quantified by qRT-PCR. LPS 3 h, WT–KO, P = 0.0000000488. d BMDMs were stimulated with recombinant mouse IFN-β for a timecourse as indicated (n = 3). F3 mRNA was quantified by qRT-PCR. e BMDMs pre-treated with DMSO, DMF, or 4-OI (1 h) before stimulation with recombinant mouse IFN-β (4 h) and harvesting cell lysates (n = 9). F3 mRNA was quantified by qRT-PCR. DMSO–4-OI, P = 0.00000018. f BMDMs (n = 4) were pre-treated with DMSO, DMF, 4-OI, or baricitinib (1 h) before LPS stimulation (3 h). F3 mRNA was quantified by qRT-PCR. DMSO–DMF, P = 0.0000144; DMSO–4-OI, P = 0.0000133; DMSO–baricitinib, P = 0.00000134. g BMDMs (n = 6) were transfected with Ctrl or Jak1 siRNA and stimulated with LPS (3 h). F3 mRNA was quantified by qRT-PCR. h Predicted transcription factor sites in the promoter of the F3 gene via the Interferome database⁴². Data presented shows the predicted ___location of transcription factors from the region spanning −1500 bp to +500 bp from the start site of the F3 gene promoter. i The ___location of STAT1- and IRF1-transcription factor sites in the promoter of the human F3 gene, analyzed from enrichment analysis of 6 independent experiments from publicly available data using the ChIP-Atlas⁴³. j Representative western blot of CASPASE-11 in BMDM cell lysates pre-treated with DMSO, DMF, or 4-OI (1 h) prior to LPS stimulation (3 h), with α-TUBULIN as loading control. Blot is representative of three independent experiments. k BMDMs (n = 3) were pre-treated with DMSO, DMF, or 4-OI (1 h) before priming with LPS (3 h) and LPS transfection (16 h). Pyroptosis is represented as percentage cell death measured by LDH release in BMDM supernatants. DMSO–10 μM DMF, P = 0.0000247; DMSO–250 μM 4-OI, P = 0.000084. l Representative western blot of TF in BMDMs pre-treated with DMSO, DMF, or 4-OI (1 h) prior to LPS priming (3 h) and LPS transfection (16 h). Blots are representative of three independent experiments. Densitometry analysis of TF in BMDM (m) supernatants and n cell lysates, with GAPDH as loading control. o–q BMDMs were pre-treated with DMSO, o DMF, or p 4-OI (1 h) before priming with LPS (3 h) and LPS transfection (16 h). Thrombin generation was measured in BMDMs in situ in plate wells using FXII-deficient plasma. q Lagtime represents time-to-clot formation. NS–DMSO, P = 0.000017; DMSO–5 μM DMF, P = 0.000044; DMSO–10 μM DMF, P = 0.0000019; DMSO–250 μM 4-OI, P = 0.00007985. Data from a–g, k are mean ± SEM from 3–6 independent experiments. Data from m–q are mean ± SD from 3 independent experiments. P values calculated using two-tailed Student’s t test for paired comparisons or one-way ANOVA for multiple comparisons. Source data are provided as a Source Data file.