Fig 2: Effects of EA at acupoints on the expression of cytochrome c, TRADD, FADD, cleaved caspase-8, and cleaved caspase-3 in the ischemic cortical penumbra.Representative western blot images show the expression of (A) mitochondrial cytochrome c and (C) cytosolic cytochrome c, TRADD, FADD, cleaved caspase-8 and cleaved caspase-3 in the ischemic cortical penumbra in the Sham-7 d, Model-7 d, EA-7 d, and Non-acup-7 d groups 7 d after reperfusion. COX IV and actin were used as internal controls for the mitochondrial and cytosolic fractions, respectively. The relative mitochondrial expression of (B) cytochrome c, and the relative cytosolic expression of (D) cytochrome c, (E) TRADD, (F) FADD, (G) cleaved caspase-8, and (H) cleaved caspase-3 were evaluated in the ischemic cortical penumbra in the Sham-7 d, Model-7 d, EA-7 d, and Non-acup-7 d groups (n = 4). Data are presented as mean ± SD. *P<0.05 compared with the Sham-7 d group; #P<0.05 compared with the Model-7 d group.

Fig 3: ORF3 suppresses NF-?B signaling via TRADD and RIP1.(A) ORF3 suppresses the phosphorylation of IKKß and I?Ba. (B) TLR3 pulls down of key downstream molecules detected by a coimmunoprecipitation assay. (C,D) TRADD and TAK1 expression levels are down-regulated in ORF3-pretreated cells compared with GFP-pretreated cells after stimulation with Poly(I:C). In contrast, TRIF, TRAF6 and RIP1 expression levels are increased by this stimulation.

Fig 4: The overexpression of TRADD restores ORF3-inhibited NF-kB signaling.(A) TRADD plasmids were co-transfected with ORF3 at a ratio of 1:2 for 48 h, and the cells were then treated with Poly(I:C) for 12 h. Phosphorylated I?Ba was assessed to measure NF-?B activity using western blotting, with ß-actin serving as a loading control. Overexpression of TRADD restores ORF3-inhibited NF-?B signaling; the relative densitometric ratios for experimental bands are shown (**P < 0.01). (B) Poly(I:C) increases the concentration of inflammatory factors (IL-8, TNF-a and IL-1ß) in the vector or TRADD groups. In presence of Poly(I:C) and ORF3, the concentration of inflammatory factors in the supernatants was lower in the vector group compared to the TRADD group (**P < 0.01). The data are given as the means ±SD representative of three independent experiments.

Fig 5: Effects of single gene knockouts on lectin-induced cell death in MC-38 cells. Intrinsic apoptosis was assessed by inactivation of BAK/BAX (A), whereas extrinsic apoptosis and necroptosis pathways were assessed by inactivation of FADD (B), TRADD (C), MLKL (D), and combined caspase 8/RIPK3 (E) genes. Pyroptosis was assessed by caspase 1 (F) and GSDMD (G) gene inactivation. To exclude clonal effects, all experiments were performed in at least two clones for each gene knockout. Data represent 6 replicates from at least 2 independent experiments. WT (blue bars), gene knockouts (red bars). *p < 0.05; unpaired two-tailed t-test.