Inhibitor of Apoptosis Proteins (IAPs) Limit RIPK1-Mediated Skin Inflammation  Holly Anderton, James A. Rickard, George A. Varigos, Najoua Lalaoui, John Silke  Journal of Investigative Dermatology  Volume 137, Issue 11, Pages 2371-2379 (November 2017) DOI: 10.1016/j.jid.2017.05.031 Copyright © 2017 The Authors Terms and Conditions

Figure 1 cIap1EKO/EKO.Xiap–(y)/– mice develop skin disease by 15 weeks of age. (a) Representative pictures of the indicated strains at the indicated ages. (b) Kaplan-Meier survival curves of cIap1EKO/EKOXiap–(y)/– alongside littermate controls. (c) Hematoxylin and eosin, immunohistochemical, and immunofluorescence analysis of ear skin sections from the indicated strains at the indicated ages. CC3 and Ki67 sections developed with DAB (brown) and counterstained with hematoxylin (blue). Toluene blue shows mast cells as dark purple. Keratin 6 is in red, and CD11B shows macrophages in green. CD3/8 shows CD8– T cells as green and CD8+ T cells as yellow/orange. Nuclei are blue. Scale bars = 50 μm. (d) Cytokine levels in ear skin lysates of the indicated strains at indicated ages. Graphs show means ± standard error of the mean, n ≥ 3; each data point represents an individual mouse. ∗P ≤ 0.05, ∗∗P ≤ 0.01, ∗∗∗∗P ≤ 0.001. CC3, Cleaved Caspase-3; EKO, epidermal knockout; H&E, hematoxylin and eosin; K6, keratin 6; TB, toluene blue; TNF, tumor necrosis factor; wk, week. Journal of Investigative Dermatology 2017 137, 2371-2379DOI: (10.1016/j.jid.2017.05.031) Copyright © 2017 The Authors Terms and Conditions

Figure 2 cIap1EKO/EKO.cIap2–/– mice develop an early widespread dermatitis. (a) Representative pictures of the indicated strains at P0, P4, and P7. Scale bars = 1 cm. (b) Kaplan-Meier survival curves of cIap1EKO/EKO.cIap2–/– alongside littermate controls. (c) Cytokine levels from dorsal skin lysates of indicated strains at E18, P0, P4, and P7. Mean ± standard error of the mean, n ≥ 3; each data point represents an individual mouse. (d) Hematoxylin and eosin, immunohistochemical, and immunofluorescence analysis of dorsal skin sections from the indicated strains at P0, P4, and P7. Scale bars = 50 μm. ∗P ≤ 0.05, ∗∗P ≤ 0.01, ∗∗∗∗P ≤ 0.001. c1, cIAP1; c2, cIAP2; CC3, Cleaved Caspase-3; E, embryonic day; EKO, epidermal knockout; H&E, hematoxylin and eosin; K6, keratin 6; P, postnatal day; TNF, tumor necrosis factor. Journal of Investigative Dermatology 2017 137, 2371-2379DOI: (10.1016/j.jid.2017.05.031) Copyright © 2017 The Authors Terms and Conditions

Figure 3 Subcutaneous injection of SM induces inflammatory skin lesions. (a) Representative pictures of SM-induced lesions in WT mice over an 11-day time course. Scale bars = 1 cm. (b) Cytokine levels in lesional lysates from WT mice treated with SM. Mean ± standard error of the mean, n ≥ 4. (c) Immunohistochemical and immunofluorescence analysis of lesional sections from Rip1+/– and WT mice. (d) Representative pictures of SM-induced skin lesions in indicated strains. Lesional areas are circled in black, injection area when lesions are absent in red. (e) CC3 staining of lesion centers in indicated strains. Scale bars in c and e = 100 μm). (f) Cytokine levels in lesional lysates from indicated strains treated with SM. Mean ± standard error of the mean, n ≥ 5. Asterisks indicate significant differences from WT at the same time point: ∗P ≤ 0.05, ∗∗P ≤ 0.01, ∗∗∗P ≤ 0.005, ∗∗∗∗P ≤ 0.001. CC3, Cleaved Caspase-3; FasL, Fas ligand; H&E, hematoxylin and eosin; ns, not significant; SM, Smac-mimetic; TNF, tumor necrosis factor; UT, untreated; WT, wild type. Journal of Investigative Dermatology 2017 137, 2371-2379DOI: (10.1016/j.jid.2017.05.031) Copyright © 2017 The Authors Terms and Conditions

Figure 4 RIPK1 heterozygosity delays onset of disease in cIap1EKO/EKO.cIap2–/– and Sharpimcpdm mice. (a) Representative pictures of the indicated strains at P0, P4, P7, and P19. Scale bars = 1 cm. (b) Kaplan-Meier survival curves of the indicated strains. cIap1EKO/EKO.cIap2–/–.Ripk1+/– data have been overlain with data from Figure 2b. (c) Cytokine levels from dorsal skin lysates from the indicated strains at E18, P0, P4, P7 and P19. Mean ± standard error of the mean, n ≥ 3’ each data point represents an individual mouse. Control and cIap1EKO/EKO.cIap–/– data as from Figure 2c. (d) Immunohistochemical and immunofluorescence analysis of dorsal skin sections, genotype, and age as indicated. Scale bars = 50 μm. (e) Representative images of indicated strains at 12 weeks of age. (F) Kaplan-Meier survival curves of indicated strains. ∗P ≤ 0.05, ∗∗P ≤ 0.01, ∗∗∗P ≤ 0.005, ∗∗∗∗P ≤ 0.001. CC3, Cleaved Caspase-3; cpdm, chronic proliferative dermatitis mutation; E, embryonic day; EKO, epidermal knockout; K6, keratin 6; K14, keratin 14; ns, not significant; P postnatal day; TNF, tumor necrosis factor. Journal of Investigative Dermatology 2017 137, 2371-2379DOI: (10.1016/j.jid.2017.05.031) Copyright © 2017 The Authors Terms and Conditions