Fig 2: GFP‐Trap immunoprecipitation of caspase‐2 BiFC dimers identifies active caspase‐2‐interacting proteins Casp2pro BiFC cells were treated with mock, 20 μM cisplatin, 50 μM etoposide, or 100 nM paclitaxel for 24 h in the presence of 10 μM Q‐VD(OMe)‐OPh, followed by GFP‐Trap IP and silver stain to examine immunoprecipitates. For a negative control, Casp2pro BiFC expression was shut off by Dox and substituted with unconjugated VN‐VC BiFC dimers pooled from cells treated similarly to experimental samples. The procedure outline is in Fig EV2A.Casp2pro BiFC dimer‐interacting proteins identified by mass spectrometric analysis were listed in descending order of peptide abundance.Casp2pro BiFC cells were treated with or without 1 μg/ml Dox for 24 h, then mock, 20 μM cisplatin, or 50 μM etoposide for 24 h in the presence of 10 μM Q‐VD(OMe)‐OPh, followed by GFP‐Trap IP and IB. BiFC fragments were detected with anti‐GFP (FL, rabbit polyclonal antibody) IB.Casp2(C320A)‐Myc and Flag‐tagged TRAF1‐7 were co‐expressed in HEK293T cells for 48 h, followed by anti‐Myc IP and IB.Casp2(C320A)‐Flag was transfected into HeLa cells and expressed for 48 h, followed by anti‐Flag IP and IB.Flag‐TRAF1‐3 were transiently expressed in HeLa cells for 48 h followed by anti‐Flag IP and IB.HeLa cells were treated with 20 μM cisplatin in the presence of 10 μM Q‐VD(OMe)‐OPh for indicated periods. Lysates were prepared and immunoprecipitated with anti‐TRAF2 antibody or control IgG, followed by IB. Source data are available online for this figure.

Fig 3: Ectopic expression of CagA increases the expression of TRAF1 and 4-1BB in GES-1 cells. (A) GES-1 cells were transfected with plasmid pEGFP-C1/CagA or control vector pEGFP-C1 for 48 h. The mRNA level of TRAF1 was determined using RT-qPCR analysis with GAPDH as the internal control. (B) GES-1 cells were transfected with plasmid pEGFP-C1/CagA or control vector pEGFP-C1 for 48 h. The mRNA level of 4-1BB was determined using RT-qPCR analysis with GAPDH as the internal control. (C) GES-1 cells were transfected with plasmid pEGFP-C1/CagA or control vector pEGFP-C1 for 48 and 72 h. TRAF1 and 4-1BB protein levels were determined using western blot analysis with GAPDH as the loading control. (D) Relative expression of TRAF1 compared with GAPDH. (E) Relative expression of 4-1BB compared with GAPDH. *P<0.05, **P<0.01, ***P<0.001. RT-qPCR, reverse transcription-quantitative polymerase chain reaction; EGFP, enhanced green fluorescent protein; CagA, cytotoxin-associated gene A; TRAF1, tumor necrosis factor receptor-associated factor 1; 4-1BB, tumor necrosis factor receptor superfamily member 9.

Fig 4: Expression of TRAF1/NF-κB p65 in vivo(A) Hematoxylin & eosin staining of human tissues from diabetic foot ulcer (DFU) wound and non-diabetes patients (NC), area framed by the rectangle is magnified, scale bar 500μm, 100 μm three samples of each group are presented.(B)The amount of p-NF-κB p65, NF-κB p65, TRAF1, and GAPDH in DFU and NC patients’ wounds measured using Western blot (n = 10, means ± SEM, t-test).∗p < 0.05, ∗∗∗p < 0.001, ns no significance.

Fig 5: Characterization of LMP1 KO effects on GM12878 LCL target gene regulation. (A) Immunoblot analysis of whole cell lysates (WCL) from GM12878 LCLs transduced with lentiviruses that express control or LMP1 targeting single guide RNAs (sgRNAs). Transduced cells were puromycin selected for 0 vs 2 days, as indicated. Blots for LMP1, for LMP1 target genes TRAF1 and IRF4, and for LMP1-driven non-canonical NF-κB pathway p100/p52 processing are shown. Blots are representative of n = 3 experiments. (B) K-means heatmap analysis of GM12878 LCLs transduced as in panel A with lentivirus expressing control or LMP1 sgRNA and puromycin selected for 48 h. The heatmap depicts relative Z-scores in each row from n = 3 independent RNAseq replicates, divided into two clusters. The Z-score scale is shown at bottom, where blue and red colors indicate lower vs higher relative expression, respectively. Two-way ANOVA P-value cutoff of <0.05 and >2-fold gene expression cutoffs were used. (C) Enrichr analysis of KEGG pathways most highly changed in GM12878 expressing control vs LMP1 sgRNA (LMP1 KO), as in panel A. The x-axis depicts the −log10 adjusted P-value (adj P-value) scale. The top three most enriched KEGG pathways are shown. (D) Abundances of two representative Cluster 1 genes from n = 3 RNAseq analyses in cells with control vs LMP1 sgRNA. P-values were determined by one-sided Fisher’s exact test. *P < 0.05, **P < 0.01. (E) Abundances of two representative Cluster 2 genes from n = 3 RNAseq analyses in cells with control vs LMP1 sgRNA. P-values were determined by one-sided Fisher’s exact test. ***P < 0.001. (F) Volcano plot analysis of host transcriptome-wide GM12878 genes differentially expressed in cells with control vs LMP1 sgRNA expression, as in panel B, using data from n = 3 RNAseq data sets. (G) Scatter plot cross comparison of log2 transformed fold change mRNA abundances in GM12878 expressing LMP1 vs control sgRNA (y-axis) vs log2 transformed fold change abundances of sgRNAs at day 21 vs day 1 post-transduction of GM12878 LCLs in a genome-wide CRISPR screen (25) (x-axis). (H) String analysis of genes shown in panel G. Pathway identifiers for each gene and interaction are colored coded. (I) Volcano plot analysis of EBV mRNA values in GM12878 expressing LMP1 vs control sgRNAs, as in panel B. P-value < 0.05 and >2-fold change mRNA abundance cutoffs were used.