Journal: Cell Death and Differentiation

Article Title: STAT1 mediates transmembrane TNF-alpha-induced formation of death-inducing signaling complex and apoptotic signaling via TNFR1

doi: 10.1038/cdd.2016.162

Figure Lengend Snippet: tmTNF-α-mediated cytotoxicity via TNFR1 was independent of DD and NSD. HEK 293T cells were transfected with empty vector, wild-type TNFR1, ΔDD-TNFR1 or ΔNSD-TNFR1 containing plasmids. (a and c) The cell surface expression of TNFR1 and its mutants (a), and TNFR2 (c) detected by flow cytometry. (b) The transfected HEK 293T cells were stimulated for 24 h with 100 ng/ml sTNF-α or tmTNF-α on fixed NIH3T3 cells at an effector/target ratio of 10:1. For neutralization of tmTNF-α, effector cells were treated with anti-TNF-α (Ab) for 30 min prior to the addition to the target cells. The cytotoxicity was detected by MTT assays. (d) HEK 293T cells were co-transfected with control or TNFR2 siRNA and expression vectors for TNFR1 or its mutants as indicated. After 48 h, the cells were stimulated with tmTNF-α on fixed NIH3T3 for 24 h. The cytotoxicity of tmTNF-α was analyzed by MTT assays. (e) The 24 h-cytotoxicity of R32W-tmTNF-α expressed on COS-7 cells towards HEK 293T cells expressing TNFR1 or mutants thereof. (f) The 24 h-cytotoxicity of sTNF-α to HEK 293T cells co-treated with indicated concentrations of S1P. (g) The 24 h-cytotoxicity of tmTNF-α to HEK 293T cells expressing TNFR1 or its mutants in the presence of 10 μM S1P. All the quantitative data represent means±S.D. of at least three independent experiments. *P<0.05, **P<0.01, ***P<0.001 versus corresponding treatment in the control group

Article Snippet: The plasmids pcDNA3.1 containing wild-type STAT1, Y701I-STAT1, S727A-STAT1 or DM (Y701I and S727A double mutations)-STAT1 were kindly gifted by Dr. Guanxin Shen (Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology). siRNA against FADD, STAT1 and TNFR2, and a scrambled control siRNA were designed and synthesized by RiboBio (Guangzhou, China).

Techniques: Transfection, Plasmid Preparation, Expressing, Flow Cytometry, Neutralization, Control

Journal: Cell Death and Differentiation

Article Title: STAT1 mediates transmembrane TNF-alpha-induced formation of death-inducing signaling complex and apoptotic signaling via TNFR1

doi: 10.1038/cdd.2016.162

Figure Lengend Snippet: tmTNF-α induced formation of DISC via TNFR1 at the plasma membrane. U937 cells were pre-treated with 100 μM MDC for 1 h and then stimulated with 100 ng/ml sTNF-α or R32W-tmTNF-α on fixed COS-7 cells at an E/T ratio of 10:1 for 30 min. For neutralization of tmTNF-α, effector cells were treated with anti-TNF-α (Ab) for 30 min prior to the addition to the target cells. The total (a), cytoplasmic (c) and membrane (d and f) protein was immunoprecipitated with an anti-TNFR1 antibody and analyzed by immunoblotting with the indicated antibodies. (e) Confocal images of the cellular distribution of FADD or caspase 8 (green) in U937 cells in response to sTNF-α or R32W-tmTNF-α (magnification, × 400). (g) Degradation of Iκ-B was analyzed by western blotting. All immunoprecipitation (IP) or/and western data are representative of three independent experiments. (b and h) The 24 h-cytotoxicity of tmTNF-α (E/T: 10:1) or sTNF-α (100 ng/ml) towards U937 cells transfected with control or FADD siRNA (b) or pre-treated with PDTC (100 μM) for 1 h (h). The data represent means±S.D. of at least three independent experiments. ***P<0.001 versus corresponding treatment in the control group

Article Snippet: The plasmids pcDNA3.1 containing wild-type STAT1, Y701I-STAT1, S727A-STAT1 or DM (Y701I and S727A double mutations)-STAT1 were kindly gifted by Dr. Guanxin Shen (Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology). siRNA against FADD, STAT1 and TNFR2, and a scrambled control siRNA were designed and synthesized by RiboBio (Guangzhou, China).

Techniques: Clinical Proteomics, Membrane, Neutralization, Immunoprecipitation, Western Blot, Transfection, Control

Journal: Cell Death and Differentiation

Article Title: STAT1 mediates transmembrane TNF-alpha-induced formation of death-inducing signaling complex and apoptotic signaling via TNFR1

doi: 10.1038/cdd.2016.162

Figure Lengend Snippet: STAT1 was necessary for tmTNF-induced DISC formation. (a and b) HEK 293T cells were transfected to express wild-type TNFR1, or ΔDD-TNFR1 and then stimulated with 100 ng/ml sTNF-α or R32W-tmTNF-α for 30 min. For neutralization, R32W-tmTNF-α-expressing cells were treated with anti-TNF-α (Ab) for 30 min prior to the addition to the target cells. The total (a) and membrane (b) protein was immunoprecipitated with an anti-TNFR1 antibody and analyzed by immunoblotting. R1: TNFR1; ΔDD: ΔDD-TNFR1. U3A (c–e) or HT1080 (f) cells were transfected by STAT1-containing plasmid or siRNA against STAT1, respectively, followed by stimulation with R32W-tmTNF-α for 30 min. Immunoprecipitation (IP)/western blotting was performed in total (c and f), membrane (d) and cytoplasmic (e) protein with an antibody to TNFR1. Ab, anti-TNF-α antibody; C, Control; T, tmTNF-α. (g) U3A cells were transfected with His-tagged empty vector, His-TNFR1 or His-ΔDD-TNFR1 containing plasmids and stimulated with R32W-tmTNF-α for 30 min. DISC formation was analyzed by IP/western blotting using an anti-His antibody. All the IP/western data are representative of three independent experiments. (h) The 24 h-cytotoxicity of R32W-tmTNF-α to U3A cells pre-treated for 30 min with caspase 8 inhibitor Z-IETD-FMK (4 μM). The data represent means±S.D. of three independent experiments. ***P<0.001 versus corresponding treatment in the control group

Article Snippet: The plasmids pcDNA3.1 containing wild-type STAT1, Y701I-STAT1, S727A-STAT1 or DM (Y701I and S727A double mutations)-STAT1 were kindly gifted by Dr. Guanxin Shen (Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology). siRNA against FADD, STAT1 and TNFR2, and a scrambled control siRNA were designed and synthesized by RiboBio (Guangzhou, China).

Techniques: Transfection, Neutralization, Expressing, Membrane, Immunoprecipitation, Western Blot, Plasmid Preparation, Control

Journal: Cell Death and Differentiation

Article Title: STAT1 mediates transmembrane TNF-alpha-induced formation of death-inducing signaling complex and apoptotic signaling via TNFR1

doi: 10.1038/cdd.2016.162

Figure Lengend Snippet: tmTNF-α-mediated cytotoxicity via TNFR1 was independent of DD and NSD. HEK 293T cells were transfected with empty vector, wild-type TNFR1, ΔDD-TNFR1 or ΔNSD-TNFR1 containing plasmids. (a and c) The cell surface expression of TNFR1 and its mutants (a), and TNFR2 (c) detected by flow cytometry. (b) The transfected HEK 293T cells were stimulated for 24 h with 100 ng/ml sTNF-α or tmTNF-α on fixed NIH3T3 cells at an effector/target ratio of 10:1. For neutralization of tmTNF-α, effector cells were treated with anti-TNF-α (Ab) for 30 min prior to the addition to the target cells. The cytotoxicity was detected by MTT assays. (d) HEK 293T cells were co-transfected with control or TNFR2 siRNA and expression vectors for TNFR1 or its mutants as indicated. After 48 h, the cells were stimulated with tmTNF-α on fixed NIH3T3 for 24 h. The cytotoxicity of tmTNF-α was analyzed by MTT assays. (e) The 24 h-cytotoxicity of R32W-tmTNF-α expressed on COS-7 cells towards HEK 293T cells expressing TNFR1 or mutants thereof. (f) The 24 h-cytotoxicity of sTNF-α to HEK 293T cells co-treated with indicated concentrations of S1P. (g) The 24 h-cytotoxicity of tmTNF-α to HEK 293T cells expressing TNFR1 or its mutants in the presence of 10 μM S1P. All the quantitative data represent means±S.D. of at least three independent experiments. *P<0.05, **P<0.01, ***P<0.001 versus corresponding treatment in the control group

Article Snippet: The plasmids pcDNA3.1 containing wild-type STAT1, Y701I-STAT1, S727A-STAT1 or DM (Y701I and S727A double mutations)-STAT1 were kindly gifted by Dr. Guanxin Shen (Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology). siRNA against FADD, STAT1 and TNFR2, and a scrambled control siRNA were designed and synthesized by RiboBio (Guangzhou, China).

Techniques: Transfection, Plasmid Preparation, Expressing, Flow Cytometry, Neutralization, Control

Journal: Cell Death and Differentiation

Article Title: STAT1 mediates transmembrane TNF-alpha-induced formation of death-inducing signaling complex and apoptotic signaling via TNFR1

doi: 10.1038/cdd.2016.162

Figure Lengend Snippet: tmTNF-α induced formation of DISC via TNFR1 at the plasma membrane. U937 cells were pre-treated with 100 μM MDC for 1 h and then stimulated with 100 ng/ml sTNF-α or R32W-tmTNF-α on fixed COS-7 cells at an E/T ratio of 10:1 for 30 min. For neutralization of tmTNF-α, effector cells were treated with anti-TNF-α (Ab) for 30 min prior to the addition to the target cells. The total (a), cytoplasmic (c) and membrane (d and f) protein was immunoprecipitated with an anti-TNFR1 antibody and analyzed by immunoblotting with the indicated antibodies. (e) Confocal images of the cellular distribution of FADD or caspase 8 (green) in U937 cells in response to sTNF-α or R32W-tmTNF-α (magnification, × 400). (g) Degradation of Iκ-B was analyzed by western blotting. All immunoprecipitation (IP) or/and western data are representative of three independent experiments. (b and h) The 24 h-cytotoxicity of tmTNF-α (E/T: 10:1) or sTNF-α (100 ng/ml) towards U937 cells transfected with control or FADD siRNA (b) or pre-treated with PDTC (100 μM) for 1 h (h). The data represent means±S.D. of at least three independent experiments. ***P<0.001 versus corresponding treatment in the control group

Article Snippet: The plasmids pcDNA3.1 containing wild-type STAT1, Y701I-STAT1, S727A-STAT1 or DM (Y701I and S727A double mutations)-STAT1 were kindly gifted by Dr. Guanxin Shen (Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology). siRNA against FADD, STAT1 and TNFR2, and a scrambled control siRNA were designed and synthesized by RiboBio (Guangzhou, China).

Techniques: Clinical Proteomics, Membrane, Neutralization, Immunoprecipitation, Western Blot, Transfection, Control

Journal: Cell Death and Differentiation

Article Title: STAT1 mediates transmembrane TNF-alpha-induced formation of death-inducing signaling complex and apoptotic signaling via TNFR1

doi: 10.1038/cdd.2016.162

Figure Lengend Snippet: STAT1 was necessary for tmTNF-induced DISC formation. (a and b) HEK 293T cells were transfected to express wild-type TNFR1, or ΔDD-TNFR1 and then stimulated with 100 ng/ml sTNF-α or R32W-tmTNF-α for 30 min. For neutralization, R32W-tmTNF-α-expressing cells were treated with anti-TNF-α (Ab) for 30 min prior to the addition to the target cells. The total (a) and membrane (b) protein was immunoprecipitated with an anti-TNFR1 antibody and analyzed by immunoblotting. R1: TNFR1; ΔDD: ΔDD-TNFR1. U3A (c–e) or HT1080 (f) cells were transfected by STAT1-containing plasmid or siRNA against STAT1, respectively, followed by stimulation with R32W-tmTNF-α for 30 min. Immunoprecipitation (IP)/western blotting was performed in total (c and f), membrane (d) and cytoplasmic (e) protein with an antibody to TNFR1. Ab, anti-TNF-α antibody; C, Control; T, tmTNF-α. (g) U3A cells were transfected with His-tagged empty vector, His-TNFR1 or His-ΔDD-TNFR1 containing plasmids and stimulated with R32W-tmTNF-α for 30 min. DISC formation was analyzed by IP/western blotting using an anti-His antibody. All the IP/western data are representative of three independent experiments. (h) The 24 h-cytotoxicity of R32W-tmTNF-α to U3A cells pre-treated for 30 min with caspase 8 inhibitor Z-IETD-FMK (4 μM). The data represent means±S.D. of three independent experiments. ***P<0.001 versus corresponding treatment in the control group

Article Snippet: The plasmids pcDNA3.1 containing wild-type STAT1, Y701I-STAT1, S727A-STAT1 or DM (Y701I and S727A double mutations)-STAT1 were kindly gifted by Dr. Guanxin Shen (Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology). siRNA against FADD, STAT1 and TNFR2, and a scrambled control siRNA were designed and synthesized by RiboBio (Guangzhou, China).

Techniques: Transfection, Neutralization, Expressing, Membrane, Immunoprecipitation, Western Blot, Plasmid Preparation, Control

Journal: Cell Death and Differentiation

Article Title: STAT1 mediates transmembrane TNF-alpha-induced formation of death-inducing signaling complex and apoptotic signaling via TNFR1

doi: 10.1038/cdd.2016.162

Figure Lengend Snippet: tmTNF-α-induced STAT1-dependent apoptotic signaling via TNFR1. sTNF-α induces binding of TRADD to the DD of TNFR1. TNFR1-bound TRADD further recruits TRAF2 and cIAP1/2 at the plasma membrane to activate NF-κB pathway. However, after TNFR1 internalization, STAT1 binds to TRADD and is phosphorylated at tyrosine 701, which excludes the binding of TRAF2 and cIAP1 to TRADD and attenuates NF-κB activation. Meanwhile, TRADD recruits FADD and caspase 8 to form DISC in the cytoplasm to mediate apoptosis. In contrast, tmTNF-α induces binding of STAT1 to the SD of TNFR1 and STAT1 serine phosphorylation at 727. The S727 phosphorylated STAT1 further recruits TRADD to form DISC at the plasma membrane to trigger apoptosis, but not NF-κB activation

Article Snippet: The plasmids pcDNA3.1 containing wild-type STAT1, Y701I-STAT1, S727A-STAT1 or DM (Y701I and S727A double mutations)-STAT1 were kindly gifted by Dr. Guanxin Shen (Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology). siRNA against FADD, STAT1 and TNFR2, and a scrambled control siRNA were designed and synthesized by RiboBio (Guangzhou, China).

Techniques: Binding Assay, Clinical Proteomics, Membrane, Activation Assay, Phospho-proteomics