Fig 2: Phenolic glycolipid (PGL)-1-mediated decrease in TRIF impairs downstream signaling events. Quantitation of mRNAs in bone marrow-derived macrophages (BMDMs) treated with 25 µM PGL-1 or vehicle (Veh) for 24 h prior to 6–24 h of stimulation with LPS/IFN-?: (A) Nos2 (iNOS); (B) Arg1 (Arginase 1); (C) IL6 (IL-6); (D) CXCL10 (CXCL10); (E) Ifnb1 (IFN-ß); (F) Tnf (TNF-a). Data are relative mean transcript levels (n > 3) ± SEM. *P < 0.05, **P < 0.01, Student’s t-test, relative to RPL19 house-keeping gene. They are representative of at least two independent experiments with similar results. Production of IFN-ß (G) and TNF-a (H) by BMDMs pretreated with 25 µM PGL-1 for 24 h prior to 6–24 h of stimulation with LPS (n = 6) or LPS/IFN-? (n = 2). Data in (G,H) are mean cytokine concentrations from biological replicates ± SEM. **P < 0.01, Mann–Whitney test comparing PGL-1-treated cells to vehicle controls.

Fig 3: JNK is recruited to necrosome and then regulates necroptosis through RIPK1 and TRIF.a, b Peritoneal macrophages were treated with TNF+zVAD (a) or poly I:C+zVAD (b) for the indicated time. Cell lysates were immunoprecipitated with anti-RIPK1 antibody and then analyzed by immunoblotting using the indicated antibodies. c Peritoneal macrophages were pretreated with zVAD, DMSO, or SP600125 for 30 min, followed by TNF, poly I:C, or LPS treatment for 3 h. Lysates were analyzed by immunoblotting with indicated antibodies. d Immunoblot analysis of peritoneal macrophages transfected with indicated siRNA for 3 days and then treated with TNF+zVAD for 3h. e Peritoneal macrophages were transfected with indicated siRNA for 3 days and then treated with TNF+zVAD and poly I:C+zVAD for 6h. Cell death was measured by released LDH. f Peritoneal macrophages transfected with control siRNA and JNK-specific siRNA for 3 days and then the expression of TLR3, TLR4, Myd88, IRAK4, A20, cyld, BIRC2, BIRC3, cFlip, JNK1, and JNK2 determined via real-time PCR. g, h Immunoblot analysis of TRIF in peritoneal macrophages (g) or MEF cells (h) transfected with indicated siRNA for 3 days. i Immunoblot analysis of TRIF in peritoneal macrophages transfected with indicated siRNA for 3 days, and then treated with TNF+zVAD or poly I:C+zVAD for 3 h. Data are representative of at least three independent experiments and shown as mean±SEM in graph e, f. *p < 0.05 by Student’s t test. NS no significance, LE long exposure, SE short exposure