flag tlr3  (Addgene inc)

Journal: The Journal of Biological Chemistry

Article Title: Surface Toll-like receptor 3 expression in metastatic intestinal epithelial cells induces inflammatory cytokine production and promotes invasiveness

doi: 10.1074/jbc.M117.784090

Figure Lengend Snippet: Poly(I:C)-responsive IECs up-regulate TLR3 expression and induce CXCL10 in a TLR3- and TRIF-dependent manner. A , TLR3 mRNA expression in SW620, SW480, HT29, HCT116, and Caco-2 cells treated with medium or poly(I:C) (5 μg/ml) for 24 h before TLR3 expression was determined by qPCR. GAPDH served as an internal control. The results show mean induction of triplicates ± S.D. relative to untreated Caco-2 cells and are representative of three independent experiments. B , TLR3 protein expression in SW620, SW480, HT29, HCT116, and Caco-2 cells left untreated (−) or treated with poly(I:C) (2.5 μg/ml) (+) for 24 h. Western blots were stained with anti-TLR3 ( top ) or GAPDH as a loading control ( bottom ). MW , molecular weight. C and D , CXCL10 release ( C ) and TLR3 mRNA expression ( D ) in SW620 cells left untreated ( 0 ), transfected with TLR3 siRNA ( siRNA TLR3.5 , 5 n m ) or NS RNA (5 n m ) or treated with the transfection reagent LF alone for 28 h prior to stimulation with poly(I:C) (2.5 or 10 μg/ml) for 20 h. CXCL10 release in the supernatant was assessed by ELISA. TLR3 mRNA expression in the lysates of the treated SW620 cells was determined by qPCR using GAPDH as an internal control. Results show mean ± S.D. of triplicates and are representative of three independent experiments. E , Western blots showing TLR3 ( top ) and GAPDH ( bottom ) expression in SW620 cells transfected with TLR3 siRNA (10 n m ), NS RNA (10 n m ), or transfection reagent (0) alone for 24 h prior to stimulation with poly(I:C) (2.5 μg/ml) for 24 h. F and G , CXCL10 production ( F ) and TRIF mRNA expression ( G ) in HT29 cells left untreated ( 0 ) or treated with siRNA against TRIF (20, 15, 10, or 5 n m ) or with NS RNA (10 n m ) for 24 h prior to stimulation with poly(I:C) (5 μg/ml) for 20 h. CXCL10 content in cell supernatant was assessed by ELISA, whereas silencing of TRIF was confirmed by assessing TRIF mRNA by qPCR using GAPDH as a reference control. The results show mean ± S.D. of triplicates and are representative of two independent experiments. ***, p < 0.001 versus NS RNA (one-way ANOVA, Holm-Sidak multiple comparisons).

Article Snippet: HEK293 cells were transfected for 24–48 h with pcDNA3 (Invitrogen) or TLR3 FLAG (Addgene, 13084) as a control for TLR3 expression using GeneJuice (Novagen) according to the instructions of the manufacturer.

Techniques: Expressing, Control, Western Blot, Staining, Molecular Weight, Transfection, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: Surface Toll-like receptor 3 expression in metastatic intestinal epithelial cells induces inflammatory cytokine production and promotes invasiveness

doi: 10.1074/jbc.M117.784090

Figure Lengend Snippet: CXCL10 production in metastatic IECs is elicited independent of poly(I:C) internalization but requires endosomal acidification. A , confocal microscopy image of HT29 cells treated with poly(I:C) Rhodamine ( red , 5 μg/ml) for 2 h before cells were washed and fixed, and the plasma membrane ( PM ) was stained with an antibody against Na,K-ATPase ( PM A488 , green ). Images ( top ) show co-localization ( Coloc , left ) and single tracks of poly(I:C) Rhodamine ( center ) and plasma membrane PM A488 staining ( right ) of the area denoted by the square in the main image. Scale bar = 5 μm. B and C , SW620 ( B ) or HT29 ( C ) cells were treated with poly(I:C) ( Added Poly(I:C) ) or double-stranded DNA dA:dT ( Added dA:dT ) added in solution or by plating cells in wells precoated with poly(I:C) ( Coated Poly(I:C) ) or dA:dT ( Coated dA:dT ) with the given concentrations for 24 h before CXCL10 release was determined by ELISA. D , CXCL10 production in HT29 cells exposed to HMW poly(I:C) or LMW poly(I:C) (2 μg/ml) either added in solution ( Added ) or by plating cells in wells precoated with poly(I:C) ( Coated ) for 21 h. E , CXCL10 release in HT29 cells pretreated with anti-TLR3 (15 or 5 μg/ml) or control goat IgG (15 μg/ml) for 1 h prior to stimulation with poly(I:C) (5 or 2 μg/ml) for 10 h. CXCL10 content in the supernatant was assessed by ELISA. **, p < 0.01; *, p < 0.05 versus cells pretreated with control IgG (two-way ANOVA, Bonferroni post-test). The results in A–D show mean ± S.D. of triplicates and are representative of three independent experiments. #, below detection. F , CXCL10 expression in HT29 cells treated with the dynamin inhibitor Dynasore (80 or 40 μ m ) or the DMSO control for 30 min prior to stimulation with poly(I:C) (2.5 μg/ml) for 8 h. CXCL10 mRNA was determined by qPCR (normalized to medium control and the endogenous control TBP). The results show the mean of triplicates from three independent experiments ± S.D. ns , not significant (two-way ANOVA, Bonferroni post-test). #, below detection. G , CXCL10 production in HT29 cells transfected with NS RNA (20 n m ) or two siRNAs against clathrin heavy chain 1 (siC2 and siC3, 10 + 10 n m ) in two rounds for 26 h and 20 h prior to stimulation with poly(I:C) (2 μg/ml) for 8 h. The results show mean ± S.D. of triplicates and are representative of three independent experiments. **, p < 0.01 versus cells transfected with non-silencing siRNA (two-way ANOVA, Bonferroni post-test). Inset , Western blots of lysates of HT29 cells treated in parallel as described for siRNAs against clathrin heavy chain 1 (siC2 or siC3) or non-silencing RNA ( N ), stained with antibody against clathrin heavy chain 1 ( CHC , top blot ) or against α-tubulin ( Tubulin , bottom blot ). H , CXCL10 production in HT29 cells pretreated with bafilomycin A (2–1 μ m ) or the DMSO control for 30 min prior to stimulation with poly(I:C) (2 or 1 μg/ml) for 10 h. The supernatant was assayed for CXCL10 content by ELISA. The results show mean ± S.D. of triplicates and are representative of two independent experiments. **, p < 0.01; *, p < 0.05 versus untreated cells (two-way ANOVA, Bonferroni post-test).

Article Snippet: HEK293 cells were transfected for 24–48 h with pcDNA3 (Invitrogen) or TLR3 FLAG (Addgene, 13084) as a control for TLR3 expression using GeneJuice (Novagen) according to the instructions of the manufacturer.

Techniques: Confocal Microscopy, Clinical Proteomics, Membrane, Staining, Enzyme-linked Immunosorbent Assay, Control, Expressing, Transfection, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Surface Toll-like receptor 3 expression in metastatic intestinal epithelial cells induces inflammatory cytokine production and promotes invasiveness

doi: 10.1074/jbc.M117.784090

Figure Lengend Snippet: TLR3 is expressed in the plasma membrane of metastatic IECs. A , Western blot of whole-cell lysate of HEK293 cells transfected with TLR3 or empty vector ( EV ) or isolated plasma membrane fractions ( PM ) or whole cell lysate ( WCL ) from HT29 cells left untreated (−) or stimulated with poly(I:C) (+) (2.5 μg/ml) for 24 h. Blots were stained with antibodies against TLR3, Na,K-ATPase as a control for the plasma membrane protein fraction, the early endosome marker EEA-1, or GAPDH. B , Western blot of whole-cell lysate of HEK293 cells transfected with TLR3 or empty vector or isolated plasma membrane fractions or whole-cell lysate of SW620 cells left untreated (−) or stimulated with poly(I:C) (2.5 μg/ml) (+) for 24 h. Blots were stained with anti-TLR3, anti-Na,K-ATPase, anti-EEA1, or anti-GAPDH. C , UNC93B1 mRNA expression in Caco-2, SW480, SW620, HCT116, and HT29 cells treated with medium or poly(I:C) (5 μg/ml) for 24 h. UNC93B1 mRNA was determined by qPCR. The results were normalized to endogenous GAPDH expression and show -fold induction relative to the Caco-2 medium sample. The results are presented as mean ± S.D. of triplicates. D and E , CXCL10 production ( D ) and UNC93B1 ( E ) expression in HT29 cells treated with siRNA against UNC93B1 (20 n m ) or NS RNA (20 n m ) twice for 26 h and 20 h prior to stimulation with poly(I:C) (2 μg/ml) for 8 h. F and G , CXCL10 production ( F ) and UNC93B1 expression ( G ) in SW620 cells treated with siRNA against UNC93B1 (20 n m ) or NS RNA (20 n m ) in two rounds for 26 h and 20 h prior to stimulation with poly(I:C) (2 μg/ml) for 24 h. D—G , CXCL10 release in the cell supernatant was assessed by ELISA, whereas UNC93B1 mRNA expression in the cells was determined by qPCR using TBP as a reference control. The results show mean ± S.D. of triplicates and are representative of three independent experiments. ***, p < 0.001; **, p < 0.01; *, p < 0.05 versus NS RNA (two-way ANOVA, Bonferroni post-test).

Article Snippet: HEK293 cells were transfected for 24–48 h with pcDNA3 (Invitrogen) or TLR3 FLAG (Addgene, 13084) as a control for TLR3 expression using GeneJuice (Novagen) according to the instructions of the manufacturer.

Techniques: Clinical Proteomics, Membrane, Western Blot, Transfection, Plasmid Preparation, Isolation, Staining, Control, Marker, Expressing, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: Surface Toll-like receptor 3 expression in metastatic intestinal epithelial cells induces inflammatory cytokine production and promotes invasiveness

doi: 10.1074/jbc.M117.784090

Figure Lengend Snippet: Poly(I:C) stimulation enhances the invasive ability of SW620 cells in a TLR3-dependent manner. A and B , SW620 and SW480 cells ( A ) or SW620 cells ( B ) transfected with silencing RNA against TLR3 (10 n m ) or NS RNA were plated in a CytoSelect 96-well invasion plate and left untreated or stimulated with poly(I:C) (10 μg/ml) for 20 h. Cells that migrated through the membrane were lysed and quantified. Results were normalized to an untreated sample. C , SW620 treated with siRNA against TLR3 or NSRNA were assayed for TLR3 mRNA by qPCR to confirm gene silencing. -Fold change is shown relative to an untreated NS RNA sample. The results show mean ± S.D. of triplicates and are representative of three independent experiments. ***, p < 0.001; **, p < 0.01 versus medium or NS RNA treatment (two-way ANOVA, Bonferroni post-test).

Article Snippet: HEK293 cells were transfected for 24–48 h with pcDNA3 (Invitrogen) or TLR3 FLAG (Addgene, 13084) as a control for TLR3 expression using GeneJuice (Novagen) according to the instructions of the manufacturer.

Techniques: Transfection, Membrane

Journal: Journal of Virology

Article Title: Disruption of MDA5-Mediated Innate Immune Responses by the 3C Proteins of Coxsackievirus A16, Coxsackievirus A6, and Enterovirus D68

doi: 10.1128/JVI.00546-17

Figure Lengend Snippet: CV-A16, CV-A6, or EV-D68 3Cpro inhibits the TLR3-mediated NF-κB response. (A) HEK293T cells were transfected with the pRL-TK plasmid, the NF-κB–luc promoter reporter, and the Flag-TLR3 expression vector alone or with CV-A16 3C or the CV-A16 3C H40D mutant. Twenty-four hours later, cells were treated with poly(I·C) for 12 h, and cell lysates were then analyzed for luciferase activity. Data are representative of results from three independent experiments. The error bars indicate the standard deviations of data from three different experiments. (B) HEK293T cells were transfected with the TAK1-Flag expression vector. Twelve hours later, cells were treated with increasing MOIs (2 and 3) of CV-A16 for 20 h. The cell lysates were then analyzed by Western blotting. (C) HEK293T cells were transfected with different amounts of HA–CV-A16 3C plasmids (lanes 2 and 3) or a control vector (lane 1). Thirty-six hours later, cells were harvested, and endogenous TAK1 and 3C-HA proteins were analyzed by Western blotting. Tubulin antibody was used as a control. (D) HEK293T cells were transfected with the pRL-TK plasmid; the NF-κB–luc promoter reporter; and the Flag-TLR3 expression vector alone or along with CV-A6 3C, EV-D68 3C, or their H40D protease mutants. Twenty-four hours later, cells were treated with poly(I·C) for 12 h, and cell lysates were then analyzed for luciferase activity. Data are representative of results from three independent experiments. The error bars indicate the standard deviations of data from three different experiments. (E and F) TAK1-Flag was transfected with CV-A6 3C or its H40D mutant (E) or with EV-D68 3C or its H40D mutant (F). Thirty-six hours later, cells were harvested, and proteins were analyzed by Western blotting. Tubulin antibody was used as a control.

Article Snippet: Flag-TLR3 was purchased from Addgene.

Techniques: Transfection, Plasmid Preparation, Expressing, Mutagenesis, Luciferase, Activity Assay, Western Blot