Journal: Scientific Reports

Article Title: Host species adaptation of TLR5 signalling and flagellin recognition

doi: 10.1038/s41598-017-17935-5

Figure Lengend Snippet: The activity of bovine and human TLR5 differs with cell background. CXCL8 protein release from ( a ) HEK293T cells and ( b ) EBL cells stably transfected with human TLR5 (♦) or bovine TLR5 (■), activated with E. coli H7 flagellin at various concentrations. The Figures show the average CXCL8 protein response for each H7 concentration as a percentage of the background response in non-transfected cells. The error bars illustrate the standard error of the mean for three experiments, each with three technical replicates. The average CXCL8 protein release in response to flagellin was significantly different in cells expressing bovine or human forms of TLR5 by ANOVA (P < 0.001) and * denotes which doses were significant by subsequent Tukey’s test (P < 0.05).

Article Snippet: Cells were passaged 48 h prior to transfection by electroporation with 2 μg of bTLR5-ptGFP1, hTLR5-ptGFP1 construct or empty ptGFP1 vector (control) in a Nucleofector™ 2d Device (Lonza Group Ltd., US) as detailed previously .

Techniques: Activity Assay, Stable Transfection, Transfection, Concentration Assay, Expressing

Journal: Scientific Reports

Article Title: Host species adaptation of TLR5 signalling and flagellin recognition

doi: 10.1038/s41598-017-17935-5

Figure Lengend Snippet: Blocking TLR signalling with down-stream inhibitors and targeted siRNA reveals similarities and differences between human and bovine signalling pathways. HEK293T cells stably transfected with human TLR5 ( a ) and EBL cells stably transfected with bovine TLR5 ( c ) were transiently transfected with siRNA for p38, RELA, TRAF6 and PIK3R1. PIK3R1 (human) and PIK3R1#b3 (bovine) are predicted to recognize all splice variants, thereby reducing levels of all three regulatory subunits of class IA PI3K encoded by human or bovine PIK3R1 respectively; PIK3R1#b2 (bovine) is predicted to only recognize the splice variant encoding the p85A regulatory subunit. In addition, cells were treated with transfection reagent only (TC) or transfected with non-target control siRNA (NTC). After 24hr cells were activated with 100 ng/ml E. coli H7 flagellin. The graphs illustrates the average release of CXCL8 protein relative to that released by NTC samples. The error bars illustrate the standard error of the mean of three experiments, each with three technical replicates. HEK293T cells stably transfected with human TLR5 ( b ) and EBL cells stably transfected with bovine TLR5 ( d ) were treated with inhibitors of p38 (5 μM SB203580), NF-κB (100 μM PDTC) and PI3K (50 μM LY294002) or DMSO before stimulation with ng/ml E. coli H7 flagellin. The graphs illustrates the average release of CXCL8 protein relative to that released by DMSO treated samples. The error bars illustrate the standard error of the mean of three experiments, each with three technical replicates. *Denotes that the average CXCL8 level was statistically significantly different from ( a and c ) NTC or ( b and d ) DMSO and H7 treated samples by ANOVA and subsequent Tukey’s test (P < 0.05).

Article Snippet: Cells were passaged 48 h prior to transfection by electroporation with 2 μg of bTLR5-ptGFP1, hTLR5-ptGFP1 construct or empty ptGFP1 vector (control) in a Nucleofector™ 2d Device (Lonza Group Ltd., US) as detailed previously .

Techniques: Blocking Assay, Stable Transfection, Transfection, Variant Assay

Journal: Scientific Reports

Article Title: Host species adaptation of TLR5 signalling and flagellin recognition

doi: 10.1038/s41598-017-17935-5

Figure Lengend Snippet: Diverse flagellin alignments of Nt and Ct D1 domains, indicating identified TLR5 binding amino acids and relative responses to these by bovine and human TLR5. ( a ) 1 Numbering according to Smith et al . (2003) . 2 Blue amino acids bind to TLR5 binding interface-B; green amino-acids bind to TLR5 binding interface-A, according to Yoon et al . (2012) ; Black amino acids bind to TLR5 according to Smith et al . (2003) . aas: amino acids. ( b ) The relative CXCL8 protein release from HEK293T cells stably transfected with human TLR5 and EBL cells stably transfected with bovine TLR5 after stimulation with 50ng/ml S . Typhimurium P1 (St P1), Listeria ivanovii FlaA (Li FlaA) and Burkholderia thailandensis FliC (Bt FliC) compared to the response to E. coli H7 (Ec H7). Error bars illustrate the standard errors of the mean for three experiments, each with three technical replicates. *Denotes that the average CXCL8 protein release in response to the different flagellin was significantly different by ANOVA and subsequent Tukey’s test (P < 0.05).

Article Snippet: Cells were passaged 48 h prior to transfection by electroporation with 2 μg of bTLR5-ptGFP1, hTLR5-ptGFP1 construct or empty ptGFP1 vector (control) in a Nucleofector™ 2d Device (Lonza Group Ltd., US) as detailed previously .

Techniques: Binding Assay, Stable Transfection, Transfection