Fig 2: Model showing how NF-?B signaling is tightly regulated by inhibitory proteins such as I?Ba and A20 (encoded by the TNFAIP3 gene) in normal nasopharyngeal epithelial cells (left), and how these proteins can be mutated in nasopharyngeal carcinoma (NPC) (right), resulting in loss of control of NF-?B signaling. We found that EBV-encoded Qp-EBNA1 can be upregulated by NF-?B, while EBNA1 protein expression has been shown to negatively regulate NF-?B activation by inhibiting IKKa/ß phosphorylation [20]. The regulatory loop between NF-?B signaling and EBNA1 implies an important role for EBV in the control of NF-?B activity in the presence of mutated genes like NFKBIA and TNFAIP3 in NPC.

Fig 3: EBV and AFB1 share a common pathway to regulate TGFBI. (a) Schematic representation of the in vitro combined AFB1-exposure and EBV-infection-mediated primary B cells immortalization experiment. (b) qPCR quantification of TGFBI mRNA expression levels in LCL obtained as explained in (a) (* p < 0.05). (c) Heatmap of CG methylation levels in B cells at different stages of EBV-mediated immortalization, in presence or absence of concomitant AFB1 in vitro exposure. Independent experiments from different B cells donors. (d) pyrosequencing-based analysis of TGFBI methylation levels within the three CpGs of interest at different time points of EBV-induced B cells immortalization with concurrent AFB1/DMSO treatment. (e) Immunoblotting quantification of Phospho-I?Ba (PIKBA) and I?Ba (IKBA) levels in Louckes vs. Louckes-EBV treated or not with AFB1 50 µM. (f) The enzymatic assays-based measure of DNMTs activity (left graph (OD/h/mg)) and DNMT1 quantity in the nucleus (right graph (ng/mg)) in RPMI-LMP1 vs. RPMI-pLXSN cell lines treated with AFB1/DMSO and with Bay11/DMSO.

Fig 4: PLA combined with RNA-FISH of the IL-1a target gene NFKBIA to study downstream NF-?B functions in the nucleus. (A) Scheme of the modified PLA procedure coupled to single molecule (sm)RNA-FISH that allows to discriminate p65/I?Ba complex formation in unresponsive cells compared to (neighbouring) cells with IL-1a-induced mRNA expression of the NFKBIA gene that encodes the I?Ba protein. (B) HeLa cells remained untreated or were stimulated for 30 or 60 min with IL-1a (10 ng/mL) as shown. Cells were fixed and p65/I?Ba complexes were revealed by PLA with specific antibodies followed by NFKBIA smRNA-FISH. PLA signals appear in green, NFKBIA FISH signals in pink and nuclei in blue (stained with Hoechst 33342). The cells were analyzed by fluorescence microscopy, representative merged pictures and PLA negative controls (from untreated cells) are shown. (C) Both, PLA and smRNA-FISH signals were counted by the Duolink®ImageTool. The box plots show the distribution of PLA or smRNA-FISH signals. The table summarizes all relevant single cell data. (D) For each cell the number of p65/I?Ba complexes was plotted against the NFKBIA smRNA-FISH signals. Data from untreated and IL-1a-stimulated conditions are depicted as separate scatter plots, red lines indicate mean signals.

Fig 5: REDD1 elicits adipocyte differentiation and macrophage inflammation through NF-?B activation.a–c Representative oil red-O (ORO)-stained images of WT and Redd1-/- SVF cells (a), shControl (shC)- or sh-Redd1-transfected 3T3-L1 cells (b), and WT (Redd1fl/fl, Rfl/fl) and Redd1?Adipoq (R?Adipoq) SVF cells (c) when cultured in differentiation medium (MDI) and quantification of relative ORO intensity (n = 4). d–f, Expression levels of adipogenic genes (d), REDD1 (e), and lipogenic genes (f) in Rfl/fl and R?Adipoq SVF cells cultured in MDI medium and quantification of relative ORO intensity (n = 4). g Assessment of NF-?B–Luc activity in 3T3-L1 cells transfected either with siRNA for control, Ikka, Ikkb, or NF-?B p65 (p65) or with pcDNA3.1/His-Ikba (n = 5). h, i Representative images and realative quantification of ORO-stained images (h) and expression levels of Pparg and Cebpa (i) in 3T3-L1 cells infected with control adenovirus (Ad-C) or adenoviral Redd1 (Ad-R) after transfection with vector alone or pcDNA3.1/His-Ikba (n = 4). j NF-?B–Luc activity in mouse peritoneal macrophages infected with Ad-C or Ad-Redd1 (n = 4). k Cytokine production in mouse peritoneal macrophages infected with Ad-C or Ad-Redd1 (n = 5). Bar graphs represent mean ± s.e.m. Statistical significance was calculated using one-way ANOVA (g, h) and two-way ANOVA (a, i) followed by the Holm–Sidak post hoc test and an unpaired two-tailed t-test (b–f, j, k). Source data are provided as a Source Data file.