Nature Immunology: doi: /ni.3866

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1 Nature Immunology: doi: /ni.3866

2 Supplementary Figure 1 The effect of TIPE2 on chemotaxis. a, The expression of TIPE2 in dhl-60c, dhl-60t, TIPE2-expressing and 15/16Q-expressing dhl-60t neutrophils was detected by Western blot. b and c, Migration tracks of individual wild type (b) and Tipe2 / (c) blood neutrophils treated with CXCL1 (200 ng/ml) on μ-slides. A total of 40 cells from each genotype were selected as described in Methods. a and b, The experiments were performed three times. The results of a representative experiment are shown. c, Live cell imaging of PtdIns(3,4,5)P 3 distribution in dhl-60c and dhl-60t neutrophils subjected to point-source stimulation with CXCL8 for 300 seconds. The PtdIns(3,4,5)P 3 distribution was visualized using the egfp-grp1-ph domain. Scale bar is 5 µm. The experiments were performed two times. The results of a representative experiment are shown. Nature Immunology: doi: /ni.3866

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4 Supplementary Figure 2 The effect of TIPE2 on the polarization of bone marrow neutrophils and dhl-60 neutrophils. a-e, The subcellular distributions of the indicated molecules in rested wild type (WT) or Tipe2 / bone marrow neutrophils (BMNs) (a-c), and WT or Tipe2 / BMNs subjected to point-source stimulation with CXCL2 (d and e) were determined by confocal microscopy. Data shown are representative images (a-d, scale bars are 5 µm) and the percentages of cells with polarized (pol) or unpolarized (unpol) distributions of F-actin (e). f-h, The subcellular distribution of F-actin, human (h) and murine (m) TIPE2 in dhl-60c, dhl-60t and mtipe2-expressing dhl-60t cells rested or subjected to point-source stimulation with CXCL8 was determined by confocal microscopy. Data shown are the percentages of cells with polarized (pol) or unpolarized (unpol) distributions of F-actin (f) and representative images (g and h, scale bars are 5 µm). a-h, The experiments were repeated three times; e and f, n 46. Nature Immunology: doi: /ni.3866

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6 Supplementary Figure 3 Quantitative measurements of the responses of wild type and Tipe2 / myeloid cells to chemoattractant stimulation. a-f, Flow cytometric analyses of actin polymerization (F-actin) (a, c and f) and AKT phosphorylation [pakt(308)] (b, d and e) in wild type (WT) and Tipe2 / bone marrow neutrophils stimulated with fmlp (a and b) or CXCL2 (c and d), and dhl-60t and TIPE2- expressing dhl-60t cells stimulated with fmlp (e and f), at the indicated times. Values represent means ± SD; *, P < The experiments were performed in triplicates and repeated three times (a-d, n=9) or two times (e and f, n=6). Nature Immunology: doi: /ni.3866

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8 Supplementary Figure 4 Selective regulation of signaling pathways by TIPE2. a, Lysates of SW480 cells transfected with control or TIPE2 plasmids were subjected to co-immunoprecipitation (co-ip) with anti-rac or control IgG. The precipitates were analyzed by Western blot for the indicated proteins. b, Lysates of SW480 cells transfected with control or TIPE2 plasmids (left panel), and wild type (WT) and Tipe2 / bone marrow-derived macrophages (BMDMs) cultured with supernatant of L929 cells (middle panel) or stimulated with CCL2 for 5 min (right panel), were subjected to pull-down with PAK-GST beads. The Rac in the pull-down (Rac-GTP) and in the lysates (total Rac) was detected by Western blot using Rac-specific antibodies. c, Lysates of WT and Tipe2 / BMDMs cultured with or without L929 cell supernatant (designated as L929 sup) or CCL2 (stimulated for 5 min) were subjected to immunoprecipitation (IP) with anti-rac or control IgG. The precipitates were analyzed by Western blot using Rac- or mtor-specific antibodies. d-f, Lysates of SW480 cells transfected with control or TIPE2 plasmids (d), WT and Tipe2 / BMDMs cultured with supernatant of L929 cells (e), and WT and Tipe2 / BMDMs treated with CCL2 for the indicated times (f) were analyzed by Western blot using antibodies to the indicated proteins. a-f, The experiments were performed at least three times. The results of a representative experiments are shown. g and h, Relative adhesion of WT and Tipe2 / BMDMs which were rested (g) or stimulated with CCL2 (h). Cell adhesion was measured at the indicated times. i, Relative adhesion of dhl-60c, dhl-60t and TIPE2-expressing dhl- 60T cells without (None) or with Rac inhibitor (Rac inh) or PI(3)K inhibitor (PI(3)K inh). For panels g-i, values represent means ± SD; *, P < 0.05; **, P < 0.01; the experiments were performed in triplicates and repeated three times (g and h, n=9) or two times (i, n=6); RU, relative units. Nature Immunology: doi: /ni.3866

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10 Supplementary Figure 5 Rac-dependent and Rac-independent functions of TIPE2 in dhl-60 neutrophils. a-d, The subcellular distribution of F-actin (a, b and d), human (h) and murine (m) TIPE2 (b-d) in dhl-60c, dhl-60t and TIPE2- expressing dhl-60t neutrophils which were stimulated with CXCL8 (point source), with or without pretreatments with Rac inhibitor (Rac inh) (a-c) or PI(3)K inhibitor (PI(3)K inh) (a, c and d). Data shown are the percentages of cells with polarized (pol) or unpolarized (unpol) distributions of F-actin and h/mtipe2 (a and c) and representative images (b and d, scale bars are 5 µm). ns, not significant. a-d, The experiments were repeated three times; a and c, n 46. Nature Immunology: doi: /ni.3866

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12 Supplementary Figure 6 Interactions between TIPE2 and phosphoinositides. a, Purified recombinant TIPE2, 15/16Q, α0-egfp, α0 15/16Q-eGFP, α0 4Q-eGFP, cofilin, and control protein trypsin inhibitor, were separated by SDS-PAGE, and stained with silver. The experiments were performed at least three times. The results of a representative experiments are shown. b, Alignment of the partial sequences of murine TNFAIP8, TIPE1, TIPE2, and TIPE3 generated by CLUSTAL W2. Identical residues are highlighted in black, and similar residues are highlighted in gray (generated by the BOXSHADE 3.21). c, The percentages of TIPE2, 15/16Q, control protein, α0-egfp, α0 15/16Q-eGFP, and α0 4Q-eGFP bound to small unilamellar vesicles containing the indicated lipids as determined by the phosphoinositide binding assay. Values represent means ± SD; **, P < 0.01; ns, not significant; the experiments were repeated at least three times (n 3). d, A schematic model of TIPE2 action in cell polarization. The plasma membrane of the neutrophil is shown in light blue; the segmented nucleus of the neutrophil is shown in light gray. e, Degree of F-actin polarization in dhl-60c, dhl-60t, TIPE2-expressing and 15/16Q-expressing dhl-60t neutrophils stimulated with CXCL8 (point source) was determined using confocal microscopy. Values represent means ± SD; *, P < 0.05; **, P < 0.01; the experiments were repeated three times; n 46. Nature Immunology: doi: /ni.3866

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14 Supplementary Figure 7 The effect of the binding of TIPE2 to phosphoinositides on cofilin-dependent depolymerization of F-actin. a-c, Time course of cofilin-dependent F-actin depolymerization performed in the presence or absence of control protein (a-c), TIPE2 (ac), small unilamellar vesicles (SUV) containing 10% PtdIns(4,5)P 2 /10% PtdIns(3,4,5)P 3 (a), SUV containing 10% PtdIns(4,5)P 2 (b) or SUV containing 10% PtdIns(3,4,5)P 3 (c). Results are shown as the difference in the remaining F-actin over the indicated period of time between samples containing control protein and samples containing control protein + SUV or TIPE2 + SUV. FIU, fluorescence intensity units. The experiments were repeated at least three times. Nature Immunology: doi: /ni.3866

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16 Supplementary Figure 8 Competent anti-mog responses of TIPE2-deficient T cells. Tipe2 / and wild type (WT) mice were immunized with myelin oligodendrocyte glycoprotein (MOG) peptide to induce experimental autoimmune encephalomyelitis (EAE). Twenty-five days after immunization, Tipe2 / and WT mice were sacrificed, and their splenocytes were isolated and cultured with the indicated amounts of MOG peptide for 24 h. Cytokine concentrations in the culture supernatants were determined by ELISA. Values represent means ± SD. *, P < 0.05; **, P < 0.01; the experiments were repeated three times, n = 8. Nature Immunology: doi: /ni.3866