Fig 2: ATG5DD stabilization inhibits ISG expression via cell-intrinsic modulation of IFN-β expression and not through desensitization to IFNAR signalling.a, b ATG5DD cells, pretreated or not with Shield1 (Sh1), were treated with recombinant mouse IFN-β at the indicated concentration for 6, 10 or 20 h, followed by surface expression analysis of CD274 (a) and H-2Kb (b) by flow cytometry. c ATG5 DD cells were pretreated with anti-IFNAR1 antibodies followed by IAV infection. Ifnb1 expression was assayed by RT–qPCR. d In independent experiments, Ifnb1 induction was measured at 1.5 and 3.5 h post-infection in autophagy-competent (Sh1) vs. autophagy-null cells (∅). e, f ATG5DD cells, pretreated or not with Shield1 (Sh1), were infected with IAV for 1 h before monitoring IκBα degradation by immunoblot (e) and quantification with ImageJ (f). g ATG5DD cells transfected with NF-κB transcription activity GFP reporter for 20 h were pretreated or not with Shield1 (Sh1) and infected with IAV. Incucyte intra-incubator microscope allowed the monitoring of GFP-positive cells. a–d, f, g Graphs show mean and standard deviation of three (a–d, f) or four (g) biological replicates, and data are representative of three experiments. ns, not significant; *q < 0.05, **q < 0.01 (one-tailed unpaired t-test followed by Holm’s multiple testing correction)

Fig 3: E0771 tumor RNA-seq. A, Number of genes showing significant upregulation or down regulation at each of five time points of metronomic CPA treatment. A total of 2,633 genes met the thresholds for a significant response (fold-change > 2 at edgeR-adjusted P < 0.05) at one or more time points (Supplementary Table S5A). B, The set of 2,633 responsive genes was classified based on the time course of response, as detailed in Supplementary Table S5B. Each set was analyzed for overlap with the set of 188 genes that were up regulated by both 4HC and IFNB in cultured E0771 cells (Fig. 3B), and enrichment scores with significance by Fisher exact text calculated compared to a background set of all genes expressed at FPKM > 1, as shown in Supplementary Table S5B.

Fig 4: 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.

Fig 5: 4-OI suppresses lung inflammation with associated coagulopathy after SARS-CoV-2 infection.a, b BMDMs were pre-treated with DMSO, DMF, or 4-OI (1 h) before poly(I:C) stimulation (3 h) and harvesting cell lysates. a F3 and b Casp4 (caspase-11) mRNA was quantified by qRT-PCR. c Representative images of H&E staining examining histological changes in lung structure and inflammatory cell infiltration in the primary airways, parenchyma, and vasculature from male K18-hACE2 mice infected with 103 PFU SARS-CoV-2 (Wuhan isolate; VIC01/2020) or mock infection and intranasally treated with PBS or 4-OI (10 mg/kg) from 1 day post infection, with daily treatments continuing until the termination of the experiment at day 6 post infection (n = 5–6 per group). d Representative images and e quantification of vWF staining in lung sections from mice treated as in c (n = 5–6 per group). Mock+PBS–SARS-CoV-2 + PBS, P = 0.00000000002; SARS-CoV-2 + PBS–SARS-CoV-2 + 4-OI, P = 0.00000000002. f Representative images and g quantification of fibrinogen staining in lung sections from mice treated as in c (n = 5, 6 per group). Mock+PBS–SARS-CoV-2 + PBS, P = 0.000016; SARS-CoV-2 + PBS–SARS-CoV-2 + 4-OI, P = 0.000002887. h Representative images and i quantification of collagen deposition in lung sections from mice treated as in c. Total numbers of j neutrophils, k lymphocytes, and l leukocytes (Mock+PBS–SARS-CoV-2 + PBS, P = 0.000078) in the bronchoalveolar lavage fluid (BALF) of mice treated as in c. Viral titers in the m BALF and in n lung homogenates from SARS-CoV-2-infected mice treated as in c, detected using plaque assays. n = 5, 6 per group for i–n. o Clinical scoring of mice (assessed for weight loss, activity level, eye closure, and appearance of fur and posture) treated as in c. Clinical scoring of mice assigned SARS-CoV-2-infected mice to both categories 2 and 3 (83% to category 2, 17% mice to category 3), while SARS-CoV-2-infected mice that received therapeutic administration of 4-OI were dispersed across no clinical score, category 1, and category 2 groups (33% mice to no clinical score, 50% mice to category 1, and 17% mice to category 2). Quantification of p Ifnb1 (Mock+PBS–SARS-CoV-2 + PBS, P = 0.00003) and q Nfe2l2 mRNA by qRT-PCR in PBMCs of mice treated as in c (n = 5, 6 per group). Human PBMCs from healthy controls (HC) (n = 5, 6) and SARS-CoV-2-infected patients (n = 9–11) were assessed for quantification of r F3 and s CASP4 mRNA by qRT-PCR. Data from a, b and r, s are mean ± SEM from three independent in vitro experiments. Data from e, g, i–n, and p, q are mean ± SD. Scale bar for c, d, f, and h = 50 μm. 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.