Fig 2: Fenofibrate re-established macrophages to enhance their antitumor function by activating the PPAR-a-STAT1 and FAO positive feedback loop pathways. (A) Immunofluorescence staining for the expression of cytokines IL-1ß and iNOS in Co-CM1, Con-CM with and without FF treatment, as well as Co-CM1 (siPPAR-a) + FF groups. (B) SIRT1, JAK1-STAT1, and FAO pathways were assessed by western blot analysis in CTRL and Co-CM1 groups. (C) ELISA of the IL-12 and IL-23 secretion by macrophages in Co-CM1 and, Con-CM with and without FF treatment. *p < 0.05, **p < 0.01. (D) Elucidation of tumor cells proliferation from culture medium of different conditioned macrophages. (E) Proliferation of MDA-MB-231 cells treated with conditioned media from different macrophages. ***p < 0.001. (F) Clone formation of MDA-MB-231 cells cultured in supernatant from different macrophages.

Fig 3: Combined immunofluorescence of HLA class I/STAT1 and PKR/VP1 in GD samples and controls. Nuclear and cytosolic STAT1 was found in thyroid cells and was colocalized with HLA class I. PKR and VP1 were also colocalized within thyroid cells. A, An example of STAT1 (green) and HLA class I (red) immunofluorescence staining in GD thyroid tissue. Arrows indicate thyroid cells with nuclear STAT1 and intracellular HLA class I. B, Control sample with negative STAT1 and HLA class I staining. C, An example of VP1 (green) and PKR (red) immunofluorescence staining in GD thyroid tissue. The insets represent higher magnification (of the area outlined by the white boxes) and shows colocalized VP1 and PKR within thyrocytes. D, Control sample with negative VP1 and PKR staining. Merged images with blue nuclear DAPI staining. All scale bars at 25 µm. DAPI, 4’,6-diamidino-2-phenylindole; GD, Graves disease; HLA, human leukocyte antigen; PKR, protein kinase R; STAT1, signal transducer and activator of transcription 1; VP1, enteroviral capsid protein 1.

Fig 4: Activation of IRF3 and augmentation of IFN-ß1 expression by VGLL3 in RA-FLS. a The mRNA expression levels of IFN-a1 and IFN-ß1 were detected by qPCR after VGLL3 overexpression or knockdown in RA-FLS. b Representative images of the expression of IRF3 in RA-FLS examined by immunofluorescence staining. Nuclei were stained with DAPI. c The protein expression levels of p-IRF3 and total IRF3 in RA-FLS were detected by Western blotting upon VGLL3 overexpression. GAPDH was used as the housekeeping gene. d The protein expression levels of p-STAT1 in RA-FLS were detected by Western blotting upon VGLL3 overexpression. ß-Tubulin was used as the housekeeping gene. e Gene expression levels of STAT1, TNFSF13B, MX1, IRF7, OAS1, TLR3, and CCL5 were detected in RA-FLS transfected with Flag-VGLL3-expressing lentivirus with/without the treatment of Janus kinase inhibitor tofacitinib (250 nM). f Representative images of the expression of STAT1 in RA-FLS transfected with Flag-VGLL3-expressing lentivirus with/without the treatment of tofacitinib (250 nM) examined by immunofluorescence staining. Nuclei were stained with DAPI. Scale bar, 100 µm. N = 3. * p < 0.05, ** p < 0.01, *** p < 0.001

Fig 5: Overexpression of Mettl3 or Mettl14 attenuates DLL-mediated IFN-? response. (a) Mettl3 and Mettl14 expression was upregulated in 3LL cells after infection with Mettl3 or Mettl14 expressing lentivirus. (b) The expression of p-Stat1 (phosphorylated), Stat1, and Irf1 was reduced by infection with Mettl3 or Mettl14 expressing lentivirus in DLL-treated 3LL cells. (c) The m6A modification of Stat1 and Irf1 was upregulated by infection with Mettl3 or Mettl14 expressing lentivirus in DLL-treated 3LL cells evaluated by m6A RIP-qPCR. (d) CLL5, CXCL9, and CXCL10 production in cell culture supernatant was reduced by infection with Mettl3 or Mettl14 expressing lentivirus in DLL-treated 3LL cells evaluated by ELISA. (e) The ROS levels were reduced by infection with Mettl3 or Mettl14 expressing lentivirus in DLL-treated 3LL cells. (f) The level of SOD was increased by infection with Mettl3 or Mettl14 expressing lentivirus in DLL-treated 3LL cells. (g) 3LL cells were cocultured for 4 days with T cells acquired from tumor-bearing mice. The specific proliferation of T cells was measured by BrdU incorporation. The proliferation of T cells was inhibited by Mettl3 or Mettl14 expressing 3LL cells, which was reversed by DLL treatment. (h) The secretion of IFN-? was detected by ELISA in cell culture supernatant that 3LL cells cocultured for 4 days with T cells acquired from tumor-bearing mice. The secretion of IFN-? was reduced by coculturing with Mettl3 or Mettl14 expressing 3LL cells, which was reversed by DLL treatment. Data are the mean ± SD from three independent experiments. *P < 0.05 and**P < 0.01 by Student's t-test, N = 6. DLL: delicaflavone.