Fig 2: A20 participates in the negative regulation of alternative NF-?B activation in H. pylori infection. a AGS cells were infected with H. pylori or stimulated with 30 ng/ml LTa1ß2. Total cell lysates were analyzed for the indicated proteins by immunoblot (IB). b AGS cells were infected with H. pylori. The cytosolic (Cyto) and soluble nuclear (N1) fractions were analyzed by IB. c Representative immunohistochemical micrographs of RelB-stained normal (upper panel) or type B gastritis (lower panel) gastric mucosal specimens. See scale bar for magnification. d Left graph shows moderate cytoplasmic RelB staining intensity in type B gastritis cases, while weaker staining intensity was observed in healthy control gastric specimens, p < 0.001. Right graph shows high nuclear RelB staining intensity in the case of type B gastritis (n = 32) compared to normal gastric mucosa (n = 41), p < 0.001. e Wild-type AGS (WT) and two clones of A20-deficient AGS cells (A20KO_1 and A20KO_2) were infected with H. pylori. Total cell lysates were analyzed for the indicated proteins by IB. f WT, A20KO_1 and A20KO_2 AGS cells were infected with H. pylori. The cytosolic (Cyto) and soluble nuclear (N1) fractions were analyzed by IB. g WT, A20KO_1 and A20KO_2 AGS cells were treated with 30 ng/ml LTa1ß2. Total cell lysates were analyzed for the indicated proteins by IB. A representative blot of at least two experiments was shown

Fig 3: USP48 controls A20 de novo synthesis and suppresses caspase cleavages. a AGS cells were transfected with siRNA against USP48 and infected with H. pylori for the indicated times. Total RNA was isolated at the indicated times and analysed using quantitative RT-PCR for the TNFAIP3 transcript (gene of A20). Data shown depict the average of triplicate determinations normalized to GAPDH housekeeping gene. Error bars denote mean ± SD. b, c AGS cells were transfected with siRNA against USP48 and infected with H. pylori for indicated times. Whole-cell lysates were subjected to IB for analysis of the indicated proteins. C-Casp8 or C-Casp3 = cleaved caspases. d AGS cells were transfected with recombinant human USP48 and infected with H. pylori for 24 h. Whole-cell lysates were subjected to IB analysis of the indicated proteins. e AGS cells were transfected with siRNA against USP48 and infected with H. pylori for the indicated times. IP with an anti-Caspase-8 antibody was performed at the indicated times in the presence of NEM and OPT, followed by IB analysis of the indicated proteins. f AGS cells transfected with siRNA were infected with H. pylori for 24 h before incubation with Caspase-Glo® 8 reagent for 1 h. Luminescence of caspase-8 activity was measured and calculated as a fold increase compared to the uninfected scramble control. g AGS cells were transfected with siRNA against USP48 and infected with H. pylori for 24 h, followed by staining with annexin V/PI. Apoptotic cell death was analysed by flow cytometry. Data shown depict the average of two independent experiments. Error bars denote mean ± SD. h AGS cells were transfected with siRNA against USP48 and infected with H. pylori for 24 h. Cleaved caspase-3/7 expression was detected by the IncuCyte® S3 Live-Cell Analysis Image system. Scale bars = 100 µm. Data shown depict the average of four pictures from distinct regions. Error bars denote mean ± SD. Data information: Data shown in (a) are from three independent experiments with three technical replicates. Data shown in (b–e, g) are representative for at least two independent experiments. Data shown in (f) are from one experiment with three technical replicates. Data shown in (h) are from one experiments with four technical replicates. *P = 0.05, **P = 0.01, ***P = 0.001 (Student’s t-test)

Fig 4: USP48-suppressed caspase cleavage is A20-dependent. a AGS cells were transfected with siRNA against A20, and subsequently transfected with recombinant human USP48. One hour after transfection, the cells were infected with H. pylori for indicated times. Whole-cell lysates were subjected to IB for analysis of the indicated proteins. b AGS cells were transfected with siRNA against A20, and subsequently transfected with recombinant human USP48. One hour after transfection, the cells were infected with H. pylori for 24 h. Cleaved caspase-3/7 expression was detected by the IncuCyte® S3 Live-Cell Analysis Image system. Scale bars = 100 µm. Data shown depict the average of four pictures from distinct regions. Error bars denote mean ± SD. c AGS cells were transfected with siRNA against USP48 and then transfected with recombinant human A20 protein. One hour after protein transfection, cells were infected with H. pylori for the indicated times. Total cell lysates were subjected to IB analysis of the indicated proteins. d Schematic representation of the findings in this study. Infection of H. pylori induces fast activation of NF-?B, leading to nuclear translocation of RelA (1) and expression of the target gene TNFAIP3 (encodes for A20) (2). Termination of RelA activity by ECSsocs1-dependent ubiquitinylation and degradation (3). CSN-associated USP48 deubiquitinylates RelA-Ub resulting in RelA stabilisation (4) and prolonged A20 de novo synthesis (5). USP48 and A20 synergistically suppresses caspase-8 activity and apoptotic cell death (6). Data information: Data shown in (a, c) are representative for at least two independent experiments. Data shown in (b) are from one experiments with four technical replicates. *P = 0.05, ***P = 0.001 (Student’s t-test)

Fig 5: Alternative NF-?B signaling contributes to cell survival in H. pylori infection. a AGS cells were transfected with non-target-specific siRNA (scr) or siRNA targeting RelB (RelBsi_E1) for 48 h. After H. pylori infection for 24 h, cells were harvested and stained with annexin V-FITC/PI, and analyzed by flow cytometry. A graph showing data from three independent experiments is depicted (means ± SD). ##p < 0.005, *p < 0.01 and **p < 0.001. Representative dot plots are shown for each treatment. b A proposed model for A20’s role in the negative regulation of alternative NF-?B signaling pathway in H. pylori infection. H. pylori-induced activation of the classical NF-?B pathway results in the up-regulation of A20 expression. A20 binds to TIFA in the TIFA/NIK regulatory complex, which in turn destabilizes the association of TIFA with the complex. This restores the stability of cIAP1 in the complex and this TIFA-free NIK regulatory complex recovers the ability to mediate the proteasomal degradation of NIK, leading to the shutdown of alternative NF-?B signaling