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. 2019 Nov 1;4(21):e131028. doi: 10.1172/jci.insight.131028

Figure 6. Reduced A20 function leads to rapid islet allograft rejection.

Figure 6

(A and B) βTC3 cells cotransfected with an NF-κB.luciferase reporter (A) or an AP-1 luciferase reporter (B) and a CMV.βgal expression construct with or without PCDNA3.1-encoding murine reference A20 or A20 with an I325N coding variant. Cells were stimulated with 200 U/mL TNF for 8 hours (A); for 5, 8, 16, and 24 hours (B); or left untreated. Data represent fold change of stimulated versus nonstimulated and 3 independent experiments. (C) WT BALB/c islets were isolated and transduced with rAd.GFP (+G), rAd.TNFAIP3 (+A) or rAd.TNFAIP3I325N (+I); incubated overnight; and stimulated with 200 U/mL TNF for 0 or 4 hours; gene expression of proinflammatory factors was assessed. Each point represents a well with 300 islets. Data cumulative over 3 independent experiments. (D) Immunoblot (IB) of A20 immunoprecipitated (IP) lysates and whole-cell lysates from βTC3 cells transfected with reference A20 or A20 I325N and IKKγ and lysed following an overnight incubation. Membranes were probed for RIPK1, IKKγ, A20, or β-actin (loading control). (E) Densitometry of coimmunoprecipitated RIPK1 compared A20 pull-down, normalized to reference A20 (WT). Data in D and E represent 2 independent experiments and 3 biological replicates. (F) Expression of TNF-induced genes in islets from Tnfaip3+/+ or Tnfaip3I325N/I325N mice stimulated with TNF for 4 hours. Data shown in cumulative with each point in a column representing an independent islet preparation. (G) Percent of diabetic recipient CBA (H2k) mice normoglycemic following transplantation of 300 homozygous I325N islets (Tnfaip3I325N/I325N (n = 8; mean survival time [MST] = 8), heterozygous I325N (Tnfaip3I325N/+) (n = 8; MST = 13), or WT (Tnfaip3+/+) (n = 8; MST = 13) islet (H2b). (H and I) Islet grafts were excised at postoperative day (POD) 10, and gene expression of known islet derived inflammatory factors (H) and nonislet derived factors (I) measured. Nontransplanted overnight-cultured isolated islets were used as baseline. Statistical significance determined by 1-way ANOVA with Tukey’s multiple comparisons post hoc test (B, C, and F), 2-tailed Student’s t test (E, H, and I), or log-rank test (G). Error bars represent ± SEM or ± SD (E), *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.001.