Extended Data Figure 3: Analysis of TNF-challenged A20 wild-type, A20 OTU(C103A), A20 ZnF4(C609A,C612A) and A20 ZnF4(Y599A,F600A) mice.

a, Body temperatures of mice in response to 300 μg TNF per kg body weight treatment. Error bars are indicated for each data point and represent the mean ± standard deviation of 3 or 4 mice per genotype. b, A heat map representing profiles of serum cytokines in 12 different genotype/TNF treatment groups. Mice (n = 3 or 4 per group) were treated for the indicated time with 300 μg TNF per kg body weight; mean values per group are represented in the heat map. Each row represents one cytokine, whose values were standardized to z-scores with a mean of zero and a standard deviation of 1, and colour-coded according to the colour key. Variances of selected serum cytokines from A20 wild-type, A20 OTU(C103A) (OTU), or A20 ZnF4(C609A,C612A) (ZnF4 Cys) mice in response to TNF stimulation were evaluated using the Student’s t-test: IL6 WT versus ZnF4 Cys 2 h P = 0.009, 4 h P = 0.030; IL6 WT versus OTU 2 h P = 0.023, 4 h P = 0.035; Cxcl1 WT versus ZnF4 Cys 2 h P = 0.011, 4 h P = 0.017; Cxcl1 WT versus OTU 2 h P = 0.047, 4 h P = 0.043; Csf3 WT versus ZnF4 Cys 4 h P = 0.017, Csf3 WT versus OTU 4 h P = 0.042; Ccl11 WT versus ZnF4 Cys 4 h P = 0.0003, Ccl11 WT versus OTU 4 h P = 0.036. c, Body temperatures of ZnF4 mutant mice in response to 300 μg TNF per kg body weight treatment. Error bars are indicated for each data point and represent the mean ± standard deviation of 4 mice per genotype. d, A heat map representing profiles of serum cytokines as in Extended Data Figure 3b, but the indicated mice (n = 4 per group) were treated for four hours with 300 μg TNF per kg body weight or PBS vehicle control. Variances of selected serum cytokines from A20 ZnF4(C609A,C612A) (ZnF4 Cys), A20 ZnF4(Y599A,F600A) (ZnF4 Ub) or the respective wild-type control mice in response to TNF stimulation were evaluated using the Student’s t-test: IL6 WT versus ZnF4 Cys P = 0.000057; IL6 WT versus ZnF4 Ub P = 0.014; Cxcl1 WT versus ZnF4 Cys P = 0.016; Cxcl1 WT versus ZnF4 Ub P = 0.012; Csf3 WT versus ZnF4 Cys P = 0.024, Csf3 WT versus ZnF4 Ub P = 0.0061; Ccl11 WT versus ZnF4 Cys P = 0.005, Ccl11 WT versus ZnF4 Ub P = 0.001. e, Analysis of myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (MOG-EAE) studies in A20 WT, A20 OTU(C103A), and A20 ZnF4(C609A,C612A) mice. Top panel, MOG-EAE disease scores over time (mean ± s.e.m.) for A20 WT (n = 15) and A20 OTU(C103A) (n = 14). A20 OTU(C103A) average daily clinical scores (ADCS) P = 0.012, Dunnett’s test versus A20 WT. Bottom panel, EAE disease scores over time (mean ± s.e.m.) for A20 WT (n = 13) and A20 ZnF4(C609A,C612A) (n = 12). A20 ZnF4(C609A,C612A) ADCS P = 0.046, Dunnett’s test versus A20 WT. f, Lower power (upper panel A) and higher power (lower panel B) microscopic images of a representative lumbar spinal cord section derived from a A20 ZnF4(C609A,C612A) mouse with a grade 3 EAE clinical score at study termination (day 30). The section is stained with haematoxylin, eosin and Luxol fast blue. A, Foci of myelinopathy and gliosis (arrows). Gr, grey matter; Wh, white matter; scale bar, 200 μm. B, Focally severe myelinopathy and gliosis (double arrows) extending from the meninges close to the grey matter (delineated by white dashed line). Scale bar, 50 μm. Data represent at least two biological replicates.