Fig 2: FABP4 regulates the JAK2/STAT2 pathway via Rap1a in macrophage inflammation.A The mRNA and protein expression levels of Rap1a, CCL8, and CXCL10 in macrophages were analyzed by qRT-PCR and western blot after transfection with Ad-GFP and Ad-FABP4 and treatment with Hcy (n = 3). B Representative immunofluorescence images and qualification of the co-localization of Rap1a (red) and F4/80 (green) in the aortic root from ApoE−/− mice. Nuclei were stained with DAPI (blue) (scale bars, 50 μm) (n = 6). C The mRNA and protein expression levels of Rap1a were examined in macrophages treated with Hcy (n = 3). D Rap1a-GTP (activated Rap1a) level was measured in macrophages transfected with Ad-FABP4 and treated with Hcy. The total Rap1a protein level was used as loading control (n = 3). E The concentrations of IL-1β, IL-6, and TNF-α were analyzed by ELISA in macrophages transfected with Ad-FABP4 and/or si-Rap1a treated with Hcy (n = 3). F The protein expression levels of FABP4, Rap1a, p-JAK2, p-STAT2, and SOCS1 were analyzed by western blot in FABP4-overexpressing macrophages transfected with si-Rap1a and/or si-JAK2 and treated with Hcy (n = 3). G The concentrations of IL-1β, IL-6, and TNF-α were examined by ELISA in macrophages described as above (n = 3). Data are expressed as the mean ± SD. *P < 0.05, **P < 0.01, versus ApoE−/− + NC, Ad-GFP + Hcy, Ad-FABP4 + Hcy, or Ad-FABP4 + si-NC + Hcy group; #P < 0.05 versus si-NC + Ad-FABP4 + Hcy group; &P < 0.05 versus Ad-FABP4 + si-JAK2 + Hcy group.

Fig 3: Role of IFN signalling in melanoma invasion plasticity. (a) Inhibition of Jak1/2 by Ruxolitinib promotes the elongated, mesenchymal phenotype of melanoma cells cultured in 3D collagen (48 h). (b) Representative image of WM3629 cells after 48 h in 3D collagen treated with DMSO or Ruxolitinib. (c) Quantification of morphology of WM3629 cells treated with IFNβ alone or IFNβ plus Ruxolitinib after 48 h in collagen. (d) Quantification of morphology of melanoma cells cultured in 3D collagen for 48 h after treatment with IFNs (overall exposure to IFNs took 96 h). (e) Representative images of WM3629 cells after 48 h in 3D collagen treated with IFNs. (f) Immunoblotting detection of Stat transcription factors Stat1, Stat2 and Stat3 activation after 1 h and 48 h IFN treatment in WM3629 cells. Scale bar 100 µm in both (b) and (e). R = round, E = elongated. p-values: *** p < 0.001, ** p < 0.01, * p < 0.05. Detailed information about Western blot can be found at Figure S4.

Fig 4: Human IFNAR2 is critical for STAT1 and STAT2 phosphorylation. The following human receptor combinations were transiently transfected into HEK293 cells: (A) VGIFNAR1 and VCIFNAR2 and (B) VGIFNAR1 and VCIFNAR2Δ347. (A,B) STAT1 and STAT2 activation was determined in transiently transfected HEK293 cells treated with 10 ng/ml Hyper-IL-6, 100 ng/ml of the synthetic cytokine ligands (GCCG, GG, CC, GC) for 30 min. Equal amounts of proteins (50 μg/lane) were analyzed via specific antibodies detecting phospho-STAT1, phospho-STAT2, STAT1, STAT2, IFNAR1 (myc-tagged), and IFNAR2 (HA-tagged). Western blot data shows one representative experiment out of three.

Fig 5: ASFV pI215L reduces the IFN-α-induced formation and nuclear accumulation of ISGF3. (A) HEK-293T cells were cotransfected with HA-tagged ASFV pI215L or its ubiquitin-conjugating activity-defective mutations (C85A, C162A, or C189A), along with Flag-tagged porcine ISGF3 complex (STAT1/STAT2/IRF9) and pISRE-Luc and pRL-TK plasmids. After 30 h, cells were harvested for luciferase assays. Data are representative of three independent experiments with n = 3 technical replicates (shown as mean ± SEM). ***, P < 0.001. (B to D) HeLa cells were transfected with HA-tagged ASFV pI215L or empty vector. At 24 h posttransfection, cells were treated with IFN-α (1,000 U/mL) for 4 h. After the cells were fixed and permeabilized, they were incubated with the corresponding primary antibodies. Alexa Fluor 488-conjugated (green) secondary antibody was used to visualize endogenous p-STAT1 (B), p-STAT2 (C), or IRF9 (D) and Cy3-conjugated (red) secondary antibody to visualize pI215L. Nuclei (blue) were stained with DAPI. Scale bar, 20 μm. (E) HEK-293T cells were transfected with HA-tagged ASFV pI215L or empty vector. After 24 h, cells were treated with IFN-α (1,000 U/mL) for 4 h and harvested for subcellular fractionation. The nuclear and cytoplasmic fractions were subjected to immunoblotting analysis. As controls of the fractionation, nuclear antibody against lamin B1 and cytoplasmic antibody against GAPDH were used.