Fig 2: Bioactivity and receptor selectivity of the TNF muteins.Mouse embryonic fibroblasts (MEF) from TNFR1-/-/TNFR2-/- mice stably transfected with the chimeric receptors TNFR1-Fas or TNFR2-Fas (A) or Kym-1 cells (B) were stimulated with wildtype human TNF (huTNF; •), scTNFR2 (?) or TNC-scTNFR2 (?). Where indicated, MEF TNFR2-Fas were pretreated with the ligand/receptor stabilizing monoclonal antibody 80M2 (open symbols; 1 µg/ml; 30 min) before TNF treatment. Cell viability was determined by crystal violet staining after 24 hours (n = 3, shown are the mean values ± SEM).

Fig 3: Qualitative (A, B: A’-R’; E) and quantitative super-resolution (C,D,F 3D-SIM) data indicating a high co-localization of PF4, TGF-ß, TNF-a (cargo markers, A’-I’) versus VAMP-8 (a-granule membrane marker) in comparison to VWF (M’-O’). PF4 was detected using goat polyclonal antibodies directed against a peptide sequence present in the N-terminal portion of human PF4 (Santa Cruz, sc-23519, Santa Cruz Biotecnology, Inc., Dallas, TX, USA), TGF-ß1/2: mouse monoclonal antibodies directed against full-length recombinant TGF-ß2 of cow origin (Santa Cruz, sc-80346, TNF-alpha: goat polyclonal antibodies directed against recombinant TNF-a of human origin (R&D Systems, AF-210-NA, R&D Systems, Minneapolis, MN, USA), VWF: rabbit polyclonal antibodies directed against human VWF (Dako, A0082, Agilent (Dako), Santa Clara, CA, USA); and LAMP-2: mouse monoclonal antibodies directed against human LAMP-2 (Developmental Studies Hybridoma Bank, H4B4, Developmental Studies Hybridoma Bank, Iowa City, IA, USA). For VAMP8 labeling, either rabbit polyclonal VAMP-8 antibodies (a gift of the S. W. Whiteheart Laboratory, University of Kentucky) or mouse monoclonal antibodies directed against a peptide present in N-terminal portion of human VAMP-8 (Santa Cruz, sc-166820) were used as appropriate for double labeling. The fluorescent donkey secondary antibodies were purchased from (Jackson ImmunoResearch, Inc., West Grove, PA, USA) Positive control (J’-L’): VAMP-8 as first antibody stained with 2 different colors of second antibody; negative control (P’-R’): VAMP-8 antibody staining versus LAMP-2 (late endosome/lysosomal membrane marker). Twenty platelets were scored for each pairing. For co-localization details, see McBride et al,3 Kamykowski et al,9 Sehgal and Storrie,17, and Rhee et al.18 The fluorescence micrographs shown are all single-plane slices from 3D-SIM image stacks. Lookup tables were chosen to show a range of intensities, with only a few granules being near intensity saturation. Control data: (D) Calculated resolution values for 3D-SIM platelet immunofluorescence. (E) Qualitative evidence for 3D-SIM resolution in Z: VAMP-8 distribution shown across a series of Z-planes. (F) The 2 VAMP-8 antibodies gave equivalent labeling as shown by fluorescent object counting. Micron bars = 2 micron. 3D-SIM, 3-dimensional–structured illumination; LAMP-2, late endosomal/lysosomal membrane protein 2; PF4, platelet factor 4; TGF-ß, transforming growth factor-ß; VAMP-8, vesicle-associated membrane protein 8; VWF, von Willebrand factor

Fig 4: Distinct TNF pathways determine the cell's fate during virus infection. a–c A549 CRISPR control (CTRL), IFNAR1 KO, and MAVS KO cells mock-infected or infected with SeV HD for 24 or 48 h. a Merged representative RNA FISH-IF images of active caspase-3 positive staining. Magnification: ×40. Scale bar = 20 µm. b Percentage of active caspase-3 positive cells within each infected culture (n = 8). c Percentage of active caspase-3 positive cells within the FL-high and DVG-high populations of each culture (n = 8). d TNFa in the supernatant of A549 CTRL, IFNAR1 KO, and MAVS KO cells mock infected or infected with SeV for 24 h. One representative experiment is shown, bar = mean ± s.e.m. of technical triplicate. e Percentage of active-caspase 3 positive MAVS KO cells infected with SeV HD for 16 h and either left un-treated (UT), treated with recombinant IFN-beta (IFNß, 100U), or treated with recombinant TNFa (10 ng/ml) for 8 h. (n = 3). f, g A549 cells were infected with SeV HD and incubated with control IgG antibody or antibodies against TNFa, TNFR1, and TNFR2 (anti-TNF Combo). f Percentage of apoptotic A549 cells from the total population (n = 4). g Percentage of active caspase-3 positive cells within FL-high and DVG-high cells (n = 4). h, i MAVSKO A549 cells were infected with SeV HD for 16 h and then incubated with recombinant TNFa at 0, 0.05, 1, and 10 ng/ml for 8 h. h. Percentage of active caspase-3 positive cells among total cells (n = 3–6). i Percentage of active caspase-3 positive staining cells within FL-high and DVG-high cells (n = 3). Data are shown as mean ± s.e.m. of fold change over UT at each TNFa dosage. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by one-way (e, h) or two-way (b, c, f, g, i) ANOVA with Bonferroni’s post hoc test. ns, non-significant. ND, non-detected. See also Supplementary Fig. 5