Tbk1-TKO! DN cells (%)! 15! 10!
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1 a! T Cells! TKO! B Cells! TKO! b! CD4! CD8! Tbk1-TKO! c! DN cells (%)! DP cells (%)! CD4 + SP cells (%)! TKO! TKO! TKO! TKO! CD8 + SP cells (%)! CD4 + T Cells (X1 6 )! d! 6-week old mice! e! 4-month old mice! 2 15! 1 5! CD8 + T Cells (X1 6 )! 1 8! 6 4! 2! CD4 + T Cells (X1 6 )! 25! 2 * 15! 1 5! * * CD8 + T Cells (X1 6 )! 15! 1 5 * * cell! Medium! α-cd3+α-cd28! f! TKO! 2 Annexin V! Supplementary Fig. 1. Thymocyte development and peripheral T-cell distribution in WT and Tbk1-TKO mice. (a) Immunoblot analysis showing the specific ablation of Tbk1 in T cells, but not in B cells, of Tbk1-TKO mice. (b,c) Flow cytometric analysis of thymocytes from WT and Tbk1-TKO mice (6 wk old). Numbers in quadrants indicate the percentage of CD4 CD8 double-negative (DN), CD4 + CD8 + double-positive (DP), CD4 + single-positive (SP), and CD8 + SP thymocytes. Data are representative plot (b) and mean ± SD values (c) of 3 mice per group. (d,e) CD4 + and CD8 + T-cell numbers from spleen, peripheral lymph nodes (pln), mesenteric lymph nodes (mln), bone marrow (BM), peripheral blood (PBL) of 6-week old mice (wt=7,ko=6) (d) and 8-month old mice (wt=4, ko=3) (e). (f) Flow cytometry analysis of apoptosis, based on annexin V staining, of WT or Tbk1-TKO CD4+ T cells incubated for 48 with medium or anti-cd3 plus anti-cd28. *P<.5;, non-significant.
2 a! WT- Tbk1- c! CD4! b! CD44! WT- CD62L! WT- Tbk1- d! CD8! Tbk1- DN cells (%)! WT-! CD4 naïve (%)! DP cells (%)! Tbk1-! WT-! *! 6 *! Tbk1-! CD4 memory (%)! 4 2 CD4 + SP cells (%)! WT-! Tbk1-! WT-! Tbk1-! WT-! CD8 + SP cells (%)! Tbk1-! WT-! Tbk1-! Supplementary Fig. 2. Inducible ablation of TBK1 in adult mice impairs T-cell homeostasis without influencing thymocyte development. (a) Immunoblot analysis of TBK1 in T cells isolated from tamoxifen-treated Tbk1 +/+ Cre-ER (WT-ER) or Tbk1 fl/fl Cre-ER (Tbk1-ER) mice (after two weeks of tamoxifen injection). (b) Flow cytometric analysis of the percentage of memory (CD44 hi CD62L lo ) and naïve (CD44 lo CD62L hi ) CD4 + T cells in the spleen of WT and Tbk1-ER mice. Data are representative plot (left) and mean ± SD values (right) of multiple mice (each circle represents a mouse). (c,d) Flow cytometric analysis of the thymocyte subpopulations from WT and Tbk1-ER mice after weeks after tamoxifen injection. Data are representative plot (c) and mean ± SD values (d). *P<.5;, non-significant.
3 Tbk1-TKO! Th IFN-γ IL-17! IFN-γ Th1! IL-17! Th17! IL-17! IFN-γ Supplementary Fig. 3. TBK1 deficiency promotes Th1 differentiation in vitro. Naive CD4 + T cells isolated from WT and Tbk1-TKO mice were stimulated for 72 hours with platebound anti-cd3 (5µg/ml) and anti-cd28 (1µg/ ml) under Th, Th1, or Th17 conditions followed by flow cytometry to measure the frequency of IFN-γ-producing Th1 cells and IL-17-producing Th17 cells
4 a! CD4 + T Cells (X1 6 )! CD4 + T Cells! CD8 + T Cells! 4! B Cells! CD8 + T Cells (X1 6 )! 1. n.d! n.d! n.d! Med LN! Lung! Med LN! Lung! Med LN! Lung! B22 + Cells (X1 6 )! 3! 2! 1! WT-ctrl! TKO-ctrl! WT-flu! TKO-flu! b! c! Ratio to initial body weight! 1.2!.9!.6!.3! Day After Infection! TKO! Cytokine (ng/ml)! 4! 3! 2! 1! TKO! IFN-γ IL-12 p4 Supplementary Fig. 4. TBK1 deficiency does not affect immune responses against influenza viral infection. Flow cytometry analysis of the indicated cell types in the mediastinal lymph nodes (Med LN) and lung (a), bodyweight loss (b), and ELISA of cytokine level in the bronchoalveolar lavage fluid (c) of WT or Tbk1-TKO (TKO) mice infected intranasally with influenza (flu) virus PR8 for 8 days. Data are presented as mean ± s.d. (n=4).
5 2. Itga4 1. Itgb1 1. Ninj1 1. cxcr ccr5 1. ccr TKO! Supplementary Fig. 5. TBK1 is dispensable for the expression of integrin. QPCR analysis of relative gene expression in freshly isolated CD4 + T cells from draining LN of WT and Tbk1-TKO mice induced with EAE for 18 days.
6 a! Total! TKO! P-p38! p38! P-ERK1/2! ERK1/2! b! Tbk1-! IKKε-! TKO! KO P- AKT! S6! c! Naïve! Memory! TKO! TKO! P- AKT! S6! d! WT-! Tbk1-! P- AKT! S6! e! Relative AKT mrna! naive TKO! memory f! HA- GFP! FLAG-! Supplementary Fig. 6. TBK1, but not IKKε, negatively regulates AKT-mTORC1 signaling. (a-c) Immunoblot (IB) analysis of the indicated phosphorylated (P-) and total proteins in freshly isolated total CD4 + T cells (a,b) or FACS sorted naïve and memory CD4+ T cells (c) from WT, Tbk1-TKO (TKO), and IKKε-KO mice. (d) IB analysis using freshly isolated CD4 + T cells of WT-ER and Tbk1-ER mice after two weeks of tamoxifen injection. (e) QPCR analysis of relative AKT expression in freshly isolated naïve and memory CD4 + T cells from WT and Tbk1-TKO mice. (f) IB analysis of the indicated proteins using HEK293T cells transfected with an empty vector or the same vector encoding FLAG-tagged TBK1 or its catalytically inactive mutant (K38A), along with vectors encoding GFP and HA-tagged AKT. A non-specific band, detected by the FLAG antibody, was indicated by an arrowhead (bottom panel).
7 a! shrna! C! #1! #2! P- S6! P-Foxo1! Foxo1! Relative mrna (fold)! b! 1. Control shrna! TBK1 shrna! * * * c! d! Migrating T Cells (X1 3 )! 25! *! 2 15! 1 5! C! sh shrna! Migrating T Cells (X1 3 )! 3 *! 2 1 C! AM! Inhibitor! e! Tbk1! 3! 2! 1! *! C! MS! Supplementary Fig. 7. TBK1 regulates AKT-mTORC1 signaling and homing gene expression in human T cells. (a) IB analysis of phosphorylated (P-) and total proteins in human PBMCs transduced with a lentiviral vector (plko.1) encoding a control luciferase shrna (C) or two different TBK1 shrnas. (b) QPCR analysis of the indicated genes using human PBMCs transduced with control (C) or TBK1 shrnas. (c,d) In vitro migration assay of activated human CD4 + T cells that were transduced with a control (C) or TBK1-specific shrna or treated with DMSO (C) and a TBK1 inhibitor, amlexanox (AM, 25 µm). The number of CD4 + T cells migrating through a human brain microvascular endothelial cell monolayer was counted as migrating T cells, and data are representative of three independent experiments performed in duplicates. (e) QPCR analysis of Tbk1 expression in human PBMCs of healthy donors (C, n=13) or MS patients (MS, n=15). *P<.5.
8 Fig. 1a! P- α-cd3 +! α-cd28! PMA + Iono! 7.5!15! 7.5!15!(min)! Fig. 1c! PMA +! Jurkat! JPM5.6! Iono (min)! 7.5!15!7.5!15! TBK1 KA! P-GST-IRF3! J.SVT35! JM4.5.2! 7.5!15!7.5!15! IKKε KA! P-GST-IRF3! Fig. 1b! PMA +! Iono (min)! TBK1 KA! P-GST-IRF3! Carma1-! KO! 7.5!15!7.5!15! NEMO-! TKO! 7.5!15!7.5!15! IKKβ- TKO! 7.5!15!7.5!15! IKK KA! P-GST-IκBα IKKε KA! P-GST-IRF3! IKK KA! P-GST-IκBα Supplementary Figure 8. Full scans of blots for figures 1a-1c.
9 Fig. 5a! TKO! P- P-Foxo1! Foxo1! P-S6K1! S6K1! Fig. 5d! Fig. 5b! P- TKO! α-cd3 + α-cd28! 7.5!15!7.5!15! min! Fig. 5e! α-cd3 +! DMSO! MRT6737! α-cd28 (h)! 1! 2! 4! 6! 1! 2! 4! 6! Fig. 5c! Fig. 5f! P- P-S6K1! S6K1! FLAG-! EAE! TKO! DMSO! MG132!! K38A!! K38A! Ub-! Fig. 5g! HA- FLAG- TBK1!+!! +!!+!!+! HA- GFP! FLAG-! Supplementary Figure 9. Full scans of blots for figures 5a-5h. Fig. 5h! FLAG-! HA- GFP! HA-
10 Fig. 5i! Rapa-! DMSO! mycin! Fig. 6d! sfig. 1a! T Cells! B Cells! Fig. 5j! P- AKT! S6! P-Foxo1! TKO! sfig. 2a! TKO! P- Foxo1! Total! sfig. 6a! TKO! P-p38! p38! P-ERK1/2! ERK1/2! sfig. 6b! Tbk1-! IKKε-! TKO! KO P- Supplementary Figure 1. Full scans of blots for figures 5i, 5j, 6d, and supplementary figures 1a and 2a.
11 sfig. 6c! P- Naïve! TKO! Memory! TKO! sfig. 6d! P- WT-! Tbk1-! sfig. 6f! sfig. 7a! P- shrna! C!#1!#2! shrna! C!#1!#2! HA- GFP! FLAG-! P-Foxo1! Foxo1! Supplementary Figure11. Full scans of blots for supplementary figures 6c, 6d, 6f, and 7a.
Regulation of T-cell activation and migration by the kinase TBK1 during neuroinflammation
Received Oct Accepted 9 Dec Published Jan DOI:.8/ncomms77 OPEN Regulation of T-cell activation and migration by the kinase TBK during neuroinflammation Jiayi Yu,, Xiaofei Zhou,, Mikyoung Chang, Mako Nakaya,,
Supplemental Figure 1
Supplemental Figure 1 1a 1c PD-1 MFI fold change 6 5 4 3 2 1 IL-1α IL-2 IL-4 IL-6 IL-1 IL-12 IL-13 IL-15 IL-17 IL-18 IL-21 IL-23 IFN-α Mut Human PD-1 promoter SBE-D 5 -GTCTG- -1.2kb SBE-P -CAGAC- -1.kb
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doi: 1.138/nature775 4 O.D. (595-655) 3 1 -ζ no antibody isotype ctrl Plated Soluble 1F6 397 7H11 Supplementary Figure 1 Soluble and plated anti- Abs induce -! signalling. B3Z cells stably expressing!
% of live splenocytes. STAT5 deletion. (open shapes) % ROSA + % floxed
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Supplementary Figures
Supplementary Figures Supplementary Figure 1. NKT ligand-loaded tumour antigen-presenting B cell- and monocyte-based vaccine induces NKT, NK and CD8 T cell responses. (A) The cytokine profiles of liver
Supplementary Figure S1 Supplementary Figure S2
Supplementary Figure S A) The blots shown in Figure B were qualified by using Gel-Pro analyzer software (Rockville, MD, USA). The ratio of LC3II/LC3I to actin was then calculated. The data are represented
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Supplementary Figures Supplementary Figure 1 DOT1L regulates the expression of epithelial and mesenchymal markers. (a) The expression levels and cellular localizations of EMT markers were confirmed by
Supplemental Figure Legends
Supplemental Figure Legends Supplemental Figure 1. SemaB / mice have normal immune cell populations. Cells were prepared from the spleens of WT and SemaB / mice, stained with various antibodies and then
Control GST GST-RAP. α2-mg. 170 kda. b-actin. 42 kda LRP-1
% of max Supplementary Figure 1 Control GST GST-RP 17 kda α2-mg 42 kda b-actin Gate: CD11c+ (DCs) Gate: F4/8+ (Mfs) IgG Cd11cCre + Lrp1 fl/fl LRP-1 Supplementary figure 1. () MDCs were pretreated with
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Supplemental Figure 1. Furin is efficiently deleted in CD4 + and CD8 + T cells. a, Western blot for furin and actin proteins in CD4cre-fur f/f and fur f/f Th1 cells. Wild-type and furin-deficient CD4 +
FIG S1 Examination of eif4b expression after virus infection. (A) A549 cells
Supplementary Figure Legends FIG S1 Examination of expression after virus infection. () 549 cells were infected with herpes simplex virus (HSV) (MOI = 1), and harvested at the indicated times, followed
well for 2 h at rt. Each dot represents an individual mouse and bar is the mean ±
Supplementary data: Control DC Blimp-1 ko DC 8 6 4 2-2 IL-1β p=.5 medium 8 6 4 2 IL-2 Medium p=.16 8 6 4 2 IL-6 medium p=.3 5 4 3 2 1-1 medium IL-1 n.s. 25 2 15 1 5 IL-12(p7) p=.15 5 IFNγ p=.65 4 3 2 1
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Supplementary Material accompanying the manuscript Interleukin 37 is a fundamental inhibitor of innate immunity Marcel F Nold, Claudia A Nold-Petry, Jarod A Zepp, Brent E Palmer, Philip Bufler & Charles
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SUPPLEMENTARY FIGURES AND TABLE Supplementary Figure S1: Characterization of IRE1α mutants. A. U87-LUC cells were transduced with the lentiviral vector containing the GFP sequence (U87-LUC Tet-ON GFP).
MATERIALS AND METHODS. Neutralizing antibodies specific to mouse Dll1, Dll4, J1 and J2 were prepared as described. 1,2 All
MATERIALS AND METHODS Antibodies (Abs), flow cytometry analysis and cell lines Neutralizing antibodies specific to mouse Dll1, Dll4, J1 and J2 were prepared as described. 1,2 All other antibodies used
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doi: 1.138/nature89 IFN- (ng ml ) 5 4 3 1 Splenocytes NS IFN- (ng ml ) 6 4 Lymph node cells NS Nfkbiz / Nfkbiz / Nfkbiz / Nfkbiz / IL- (ng ml ) 3 1 Splenocytes IL- (ng ml ) 1 8 6 4 *** ** Lymph node cells
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and follicular helper T cells is Egr2-dependent. (a) Diagrammatic representation of the
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Nature Genetics: doi: /ng Supplementary Figure 1
Supplementary Figure 1 MSI2 interactors are associated with the riboproteome and are functionally relevant. (a) Coomassie blue staining of FLAG-MSI2 immunoprecipitated complexes. (b) GO analysis of MSI2-interacting
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Supplementary Figures
Supplementary Figures Supplementary Figure 1 Characterization of stable expression of GlucB and sshbira in the CT26 cell line (a) Live cell imaging of stable CT26 cells expressing green fluorescent protein
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www.sciencesignaling.org/cgi/content/full/3/114/ra23/dc1 Supplementary Materials for Regulation of Zap70 Expression During Thymocyte Development Enables Temporal Separation of CD4 and CD8 Repertoire Selection
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MiR-29 controls innate and adaptive immune responses against intracellular bacterial infection by targeting IFN-γ Feng Ma 1,2,5, Sheng Xu 1,5, Xingguang Liu 1, Qian Zhang 1, Xiongfei Xu 1, Mofang Liu 3,
SUPPLEMENTARY INFORMATION. Supp. Fig. 1. Autoimmunity. Tolerance APC APC. T cell. T cell. doi: /nature06253 ICOS ICOS TCR CD28 TCR CD28
Supp. Fig. 1 a APC b APC ICOS ICOS TCR CD28 mir P TCR CD28 P T cell Tolerance Roquin WT SG Icos mrna T cell Autoimmunity Roquin M199R SG Icos mrna www.nature.com/nature 1 Supp. Fig. 2 CD4 + CD44 low CD4
of whole cell cultures in U-bottomed wells of a 96-well plate are shown. 2
Supplementary online material Supplementary figure legends Supplementary Figure 1 Exposure to T reg cells causes loss of T resp cells in co-cultures. T resp cells were stimulated with CD3+CD28 alone or
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Supplementary Figure 1 DNA-methylation machinery is essential for silencing of Cd4 in cytotoxic T cells. (a) Scheme for the retroviral shrna screen. (b) Histogram showing CD4 expression (MFI) in WT cytotoxic
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SUPPLEMENTARY MATERIAL IL-1 signaling modulates activation of STAT transcription factors to antagonize retinoic acid signaling and control the T H 17 cell it reg cell balance Rajatava Basu 1,5, Sarah K.