Fig 2: 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 3: Inhibition of H. pylori-mediated alternative NF-?B signaling pathway by A20 requires TIFA. a WT and A20KO_1 AGS cells were infected with H. pylori. Total cell lysates were harvested and IP was performed using an antibody against TRAF2 or isotype-matched IgG (IgG). The numbers indicate the band intensities of cIAP1 (normalized to the respective band intensities of TRAF2) of H. pylori-infected samples relative to uninfected control. b WT and TIFA-knockout (TIFAKO) AGS cells were infected with H. pylori or treated with 30 ng/ml LTa1ß2. Total cell lysates were subjected to IP as in (a). c AGS cells were transfected with non-target-specific siRNA (scr) or siRNAs targeting TRAF2 (TRAF2si) or cIAP1 (cIAP1si_8) for 48 h prior to infection with H. pylori. Total cell lysates were used for IP with an antibody against TIFA or isotype-matched IgG. d Following incubation of recombinant human A20 and TIFA proteins in vitro, IP was performed using an antibody against TIFA. e as in (d) but IP was performed using an antibody against A20 or isotype-matched IgG. f A20KO_1 AGS cells were infected with H. pylori and IP was performed as in (c). IB analyses were performed with TIFA-immunoprecipitates without and with one hour incubation with 100 ng recombinant A20 protein prior to elution of immunoprecipitates. A representative blot of at least two experiments was shown

Fig 4: Adiponectin receptor agonist adiporon induces a20 expression in explanted fibroblasts and in mice.A, B Normal skin fibroblasts transiently transfected with A20-luc (A) or an NF-?B binding site mutated A20 (B) promoter-luc were incubated with AdipoRon (10 µg/ml) for indicated periods (A) or for 4 h (B), and whole-cell lysates were assayed for their luciferase activities. Results, normalized with Renilla luciferase (n = 3), shown as mean ± SD of triplicate determinations. C Left panels: Fibroblasts were incubated with AdipoRon (10 µg/ml) for indicated periods, and whole-cell lysates were examined by immunoblotting. Representative immunoblots (n = 2). A20 levels, normalized for GAPDH, are shown below each lane. Right panels: mouse embryonic fibroblasts from wild-type and A20-/- mice were treated with AdipoRon (10 µg/ml), and whole-cell lysates were examined by immunoblotting (n = 2). D C57Bl/6 mice were treated with bleomycin (200 µg/mouse) or PBS along with oral AdipoRon (50 mg/kg) given daily mice were sacrificed at day 22, and lesional skin was harvested for immunolabeling with antibodies to A20 (n = 3). E Left panels: representative images. Scale bar = 100 µm. Right panel: proportion of immunopositive interstitial cells within the dermis determined at four randomly selected locations/hpf. Results are mean ± SD (n = 3). One-way ANOVA followed by Sidak’s multiple comparison test.

Fig 5: MALT1 proteolytic activity is not essential for constitutive NF-?B, JNK and Akt signalling in PCa cells, but contributes to the expression of cancer-related proteins. (A) PCa cell lines were treated with MALT1 (1 µM) or PKC inhibitor (10 µM) for 48 h (+ indicates each type of inhibitors applied to each cell line). CYLD and A20 processing, as well as c-Jun, I?Ba, and Akt phosphorylation, were analysed by immunoblotting using the corresponding phospho-specific antibodies. (B) PCa cell lines were pre-treated with MALT1 inhibitor (2 µM) for 48 h, after which the media were refreshed with fresh inhibitor and analyzed for IL-6 expression in the supernatant by ELISA after additional 48 h. (C) PC3 MALT1 KO cells were reconstituted with either wild type MALT1 (WT) or catalytically inactive MALT1 (C/A), as indicated. CYLD cleavage was visualised by western blot of the corresponding cell extracts. Cells were treated with Brefeldin A. After 6 h, expression of 84 human cancer-related proteins was analyzed via a multiplex antibody array. Quantification was conducted using ImageJ 1.53 software [45] and is shown in the bottom panel for a selected number of proteins (marked with red boxes). Results (A,B) are representative of three independent experiments. Error bars (B) represent means ± SEs.