Journal: BMC Molecular Biology

Article Title: Preamplification techniques for real-time RT-PCR analyses of endomyocardial biopsies

doi: 10.1186/1471-2199-9-3

Figure Lengend Snippet: ABI inventoried Taqman ® gene expression assays

Article Snippet: TLR8 , Hs00152972_m1.

Techniques: Gene Expression

Journal: Nature chemical biology

Article Title: Small-molecule inhibition of TLR8 through stabilization of its resting state

doi: 10.1038/nchembio.2518

Figure Lengend Snippet: (a) Chemical structures of CU-CPT8m and 6 (negative control), concentration-response curve and dose-dependent cytotoxicity of CU-CPT8m in HEK-Blue TLR8 cell line. Data was normalized to a DMSO control (data are mean ± SD ; n = 3 independent experiments). (b) ITC thermogram of CU-CPT8m titrated into hTLR8 to determine binding affinity and stoichiometry (representative of one independent experiment). The raw data are presented on top and the integrated peak areas are shown and fitted below. Mean K d = 0.22 μM; stoichiometric binding N = 0.5. (c) Specificity test for CU-CPT8m (1 μM) with TLR-specific agonists used to selectively activate different HEK-Blue TLR-overexpressing cells in the presence or absence of 1 μM CU-CPT8m (data are mean ± SD ; n = 3 independent experiments). (d) TNF-α and IL-8 mRNA level in R848 treated HEK-Blue TLR8 cells in the presence and absence of 1 μM CU-CPT8m or the negative control, 6 (10 μM). Data are the average quantification of two independent experiments. (e) Dose-dependent response of CU-CPT8m on TLR8-mediated TNF-α production in THP-1 cells with indicated concentration of CU-CPT8m or 6 . Data are mean ± SD ; n = 3 independent experiments. (f) Dose-dependent response of CU-CPT8m or 6 on TLR8-mediated TNF-α production in PBMC cells induced by 1 μg/mL R848. Data are mean ± SD ; n = 3 independent experiments.

Article Snippet: TLR8 antibody (Novus Biologicals; NBP2-24972) was added in PBS containing 1% FBS, then incubated for 10 min.

Techniques: Negative Control, Concentration Assay, Control, Binding Assay

Journal: Nature chemical biology

Article Title: Small-molecule inhibition of TLR8 through stabilization of its resting state

doi: 10.1038/nchembio.2518

Figure Lengend Snippet: (a) Front (top) and side (bottom) views of the unliganded (left, PDB ID 3W3G), TLR8/ CU-CPT8m (middle) and TLR8/R848 (right, PDB ID 3W3N) complexes. TLR8 and its dimerization partner TLR8* are colored green and cyan, respectively. The distances between the C-termini of the two protomers of TLR8 dimer (TLR8/ CU-CPT8m ) is similar to that of the unliganded dimer (right). Superimposition of the TLR8 structure complexed with CU-CPT8m onto the corresponding unliganded TLR8 segment (a.a. 32–816) produces root-mean-square deviation (RMSD) values of 2.4 Å. The ligand molecules are illustrated by space-filling representations. The C, O and N atoms of the ligands are colored yellow, red, and blue, respectively. (b) Close-up view of antagonist binding site of unliganded TLR8 (left) and TLR8/ CU-CPT8m (right). Water molecules are indicated by red filled circles. (c) Schematic representation of interactions between CU-CPT8m and the TLR8 protein. The hydrophobic pocket and hydrogen bonds are shown as dashed gray arcs and dashed red lines, respectively.

Article Snippet: TLR8 antibody (Novus Biologicals; NBP2-24972) was added in PBS containing 1% FBS, then incubated for 10 min.

Techniques: Binding Assay

Journal: Nature chemical biology

Article Title: Small-molecule inhibition of TLR8 through stabilization of its resting state

doi: 10.1038/nchembio.2518

Figure Lengend Snippet: LRR8, LRR11-13, LRR15-16, and LRR17-18 are colored yellow, green, blue, and purple, respectively. In the bottom panel, the antagonist and agonist are illustrated by yellow and orange circles. Interactions between ligands and protruding loop regions are shown by dashed arrows. TLR8 utilized LRR11-13 in common for both agonist and antagonist binding on one side of the interface, while on the other side LRR17*-18* and LRR15*-16* for agonist and antagonist binding, respectively. Binding of agonist (e.g. R848 ) brings two TLR8 C-termini to a closer distance to initiate downstream signaling; while binding of antagonists (e.g. CU-CPT8m , CU-CPT9b ) at the antagonist binding site stabilizes inactive TLR8 dimer with C-termini further apart, preventing TLR8 from activation.

Article Snippet: TLR8 antibody (Novus Biologicals; NBP2-24972) was added in PBS containing 1% FBS, then incubated for 10 min.

Techniques: Binding Assay, Activation Assay

Journal: Nature chemical biology

Article Title: Small-molecule inhibition of TLR8 through stabilization of its resting state

doi: 10.1038/nchembio.2518

Figure Lengend Snippet: (a) Chemical structure of CU-CPT9a and CU-CPT9b. (b) Close-up view of antagonist binding site (left) and its schematic representation of TLR8/ CU-CPT9b (right). The C, O and N atoms of the ligands are colored yellow, red, and blue, respectively. Water molecules mediating the ligand recognition are indicated by red filled circles and hydrogen bonds by dashed lines. (c) Dose-dependent dimerization of TLR8. Elution profiles of gel filtration chromatography of TLR8 with CU-CPT9b (left) and R848 (right) at various concentrations. Retention volume and normalized absorbance at 280 nm (A 280 ) are shown on the left, and retention volume of TLR8 peak is plotted against its molar ratio (ligand/TLR8) on the right (representative of one independent experiment).

Article Snippet: TLR8 antibody (Novus Biologicals; NBP2-24972) was added in PBS containing 1% FBS, then incubated for 10 min.

Techniques: Binding Assay, Filtration, Chromatography

Journal: The Journal of Clinical Investigation

Article Title: p53-responsive TLR8 SNP enhances human innate immune response to respiratory syncytial virus

doi: 10.1172/JCI128626

Figure Lengend Snippet: (A) Graphical location of TLR8 p53-SNP rs3761624 (A/G*) relative to the TSS of the gene and to the p53 Response Element (p53RE). Blinded TLR8 gene (B) and protein (C) expression and (D) p53 occupancy profiles in human lymphocytes after 24 hours of treatment with p53 activators nutlin (10 μM), DXR (1 μM), and IR (4 Gy). Each dot represents a different donor. A total of 27 donors were evaluated for gene and protein expression, and 17 for occupancy. Presented in (E) nutlin, (F) DXR, and (G) IR are the decoded 24-hour results for TLR8 mRNA (n = 25) and protein (n = 25) expression profiles and p53 occupancy (n = 16) grouped by rs3761624 A/G genotypes. The horizontal bars represent the mean values. *P < 0.05; **P < 0.01; ***P < 0.0001 (2-tailed unpaired Student’s t test).

Article Snippet: A subset of samples was genotyped for the TLR8 rs rs3761624 SNP using the TaqMan SNP genotyping assay C__27497635_10 by the EPR laboratory.

Techniques: Expressing

Journal: The Journal of Clinical Investigation

Article Title: p53-responsive TLR8 SNP enhances human innate immune response to respiratory syncytial virus

doi: 10.1172/JCI128626

Figure Lengend Snippet: (A) IL-6 secretion measured by ELISA in blinded samples from PHA-stimulated lymphocytes pretreated with DMSO, nutlin, DXR, or IR for 24 hours then washed twice with PBS and challenged with water, TLR8 PAMP ssRNA40 (5 μg/mL) or its decoy ssRNA41 (5 μg/mL) for 3 hours. Each dot represents a different donor (n = 27). Presented are IL-6 secretion profiles (n = 25) grouped by A/G rs3761624 genotypes after (B) nutlin, (C) DXR, and (D) IR treatments for 24 hours. #P < 0.001 for drug-treated samples when compared with NT or DMSO treatments after ssRNA40 challenge for each genotype (2-tailed unpaired Student’s t test). *P < 0.01; **P < 0.001; ***P < 0.0001 when compared with drug- and ssRNA40-treated group expressing the A (male) or AA (female) rs3761624 SNP allele (s) (Wilcoxon signed-rank test).

Article Snippet: A subset of samples was genotyped for the TLR8 rs rs3761624 SNP using the TaqMan SNP genotyping assay C__27497635_10 by the EPR laboratory.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing

Journal: The Journal of Clinical Investigation

Article Title: p53-responsive TLR8 SNP enhances human innate immune response to respiratory syncytial virus

doi: 10.1172/JCI128626

Figure Lengend Snippet: DNA was isolated from whole blood, and infants with mild or severe RSV infection were genotyped for TLR8 rs3761624 SNP. χ2 analysis of RSV disease severity among (A) female and (B) male homozygous WT, heterozygous, and homozygous variants was used to determine association of genotype with increased disease severity. The percentage of patients for each genotype is shown below each bar.

Article Snippet: A subset of samples was genotyped for the TLR8 rs rs3761624 SNP using the TaqMan SNP genotyping assay C__27497635_10 by the EPR laboratory.

Techniques: Isolation, Infection

Journal: International Journal of Oncology

Article Title: TLR7 and TLR8 expression increases tumor cell proliferation and promotes chemoresistance in human pancreatic cancer

doi: 10.3892/ijo.2015.3069

Figure Lengend Snippet: Detection of TLR7 and TLR8 expression in pancreatic cancer, chronic pancreatitis and normal pancreatic tissue. (A) In immunohistochemical staining, strong expression (UICC II) and very strong expression of TLR7 and TLR8 (UICC III) was observed in pancreatic cancer. Increased expression of TLR7 and TLR8 was detected in chronic pancreatitis. No or occasionally low expression in normal pancreas was observed. DAB (3,3′-diaminobenzidine) brown color, Haemalaun blue color for nuclear counterstaining. Original magnification, ×100 and ×200. (B) Immunohistochemical expression of TLR7 and TLR8 in normal pancreatic tissue, chronic pancreatitis, pancreatic cancer from UICC II and UICC III patients. All error bars of immunohistochemical results represent standard error of the mean. (C) Significant gene expression of TLR7 and TLR8 in advanced tumor stages (UICC III, ** P<0.001). Increased gene expression of TLR7 and TLR8 in low tumor stages (UICC II) and chronic pancreatitis. Normal pancreatic tissue was standardized to baseline. The relative gene expression is expressed as 2 −ΔΔCq . (D) Confirmation of increased TLR7 and TLR8 protein expression in pancreatic cancer (UICC III) compared to normal pancreatic tissue by western blot analysis. β-actin probe was used as a control for protein loading.

Article Snippet: Blots were probed with antibodies to TLR7 (ProSci), TLR8 (ProSci), β-actin (Santa Cruz Biotechnology) and COX-2 (Santa Cruz Biotechnology and Novus Biologicals LLC, Littleton, CO, USA).

Techniques: Expressing, Immunohistochemical staining, Staining, Gene Expression, Western Blot, Control

Journal: International Journal of Oncology

Article Title: TLR7 and TLR8 expression increases tumor cell proliferation and promotes chemoresistance in human pancreatic cancer

doi: 10.3892/ijo.2015.3069

Figure Lengend Snippet: Detection of TLR7 and TLR8 expression in dissociated pancreatic cancer cells, chronic pancreatitis and normal pancreatic cells by FACS analysis. (A) In normal pancreatic cells no positivity for TLR7 and CD34 was shown. Little expression of TLR8 was observed. (B) Cells derived from chronic pancreatitis showed increased expression of TLR7 and TLR8 but not of CD34. (C) In pancreatic cancer cells (UICC III) elevated expression levels of TLR7, TLR8 and CD34 were demonstrated. TLR7 blue line, TLR8 red line, CD34 purple line and IgG control black line. One representative experiment of three.

Article Snippet: Blots were probed with antibodies to TLR7 (ProSci), TLR8 (ProSci), β-actin (Santa Cruz Biotechnology) and COX-2 (Santa Cruz Biotechnology and Novus Biologicals LLC, Littleton, CO, USA).

Techniques: Expressing, Derivative Assay, Control

Journal: International Journal of Oncology

Article Title: TLR7 and TLR8 expression increases tumor cell proliferation and promotes chemoresistance in human pancreatic cancer

doi: 10.3892/ijo.2015.3069

Figure Lengend Snippet: Immunohistochemical detection of TLR7 and TLR8 on cytospin samples of dissociated pancreatic cancer cells. (A) Increased expression of TLR7, TLR8 and CD34 was detected (arrows) in pancreatic cancer cells (UICC III). Elevated levels of TLR7 and TLR8 were also found in pancreatic cells from chronic pancreatitis while CD34 was not or sporadic detectable (arrows). No positivity of TLR7 and CD34 was detected in normal pancreatic cells, little expression of TLR8 was observed (arrow). DAB (3,3′-diaminobenzidine) brown color, Haemalaun blue color for nuclear counterstaining. (B) Immunofluorescence double staining in pancreatic cancer cells (UICC III, cytospins) showed increased coexpression (merge) of CD34 (FITC green and DAPI blue for nuclear counterstaining) with TLR7 (Cy3 red and DAPI blue for nuclear counterstaining) and TLR8 (Cy3 red and DAPI blue for nuclear counterstaining) (arrows). FITC, fluorescein isothiocyanate; DAPI, 4′,6-Diamidino-2-phenylindoledihydrochloride.

Article Snippet: Blots were probed with antibodies to TLR7 (ProSci), TLR8 (ProSci), β-actin (Santa Cruz Biotechnology) and COX-2 (Santa Cruz Biotechnology and Novus Biologicals LLC, Littleton, CO, USA).

Techniques: Immunohistochemical staining, Expressing, Immunofluorescence, Double Staining

Journal: International Journal of Oncology

Article Title: TLR7 and TLR8 expression increases tumor cell proliferation and promotes chemoresistance in human pancreatic cancer

doi: 10.3892/ijo.2015.3069

Figure Lengend Snippet: Depiction of the successful transduction of TLR7 and TLR8 in PANC 1 cells. (A) Increased gene expression of TLR7 and TLR8 was detected in TLR7 + and TLR8 + PANC1 cells by RT-qPCR compared to empty vector PANC1 cells. Empty vector PANC1 cells were standardized to baseline. The relative gene expression is expressed as 2 −ΔΔCq ; * P<0.0001, ** P<0.005. (B) Agarose gel electrophoresis of RT-qPCR products. PANC1 cells transduced with empty vector and peripheral blood mononuclear cells (PBMCs) were used as controls. β-actin probe was used as internal control for RT-qPCR. (C) Confirmation of increased TLR7 and TLR8 protein expression in transduced PANC1 cells by western blot analysis. PANC1 cells transduced with empty vector were used as controls. β-actin probe was used as a control for protein loading.

Article Snippet: Blots were probed with antibodies to TLR7 (ProSci), TLR8 (ProSci), β-actin (Santa Cruz Biotechnology) and COX-2 (Santa Cruz Biotechnology and Novus Biologicals LLC, Littleton, CO, USA).

Techniques: Transduction, Gene Expression, Quantitative RT-PCR, Plasmid Preparation, Agarose Gel Electrophoresis, Control, Expressing, Western Blot

Journal: International Journal of Oncology

Article Title: TLR7 and TLR8 expression increases tumor cell proliferation and promotes chemoresistance in human pancreatic cancer

doi: 10.3892/ijo.2015.3069

Figure Lengend Snippet: Expression and stimulation of TLR7 and TLR8 causes increased proliferation in TLR7 + and TLR8 + PANC1 cells. (A) Increased tumor size in subcutaneously injected Balb/c nude mice triggered by TLR7 + (n=5) and TLR8 + (n=5) PANC1 cells compared to empty vector PANC1 cells (n=4). (B) Significant increase in tumor volume caused by TLR7 + PANC1 cells in Balb/c nude mice compared to empty vector cells ( ** P<0.005). (C) Significantly accelerated proliferation of TLR7 + and TLR8 + PANC1 cells without ( ** P<0.002 and ** P<0.005) and with R848 stimulation ( *** P<0.02 and * P<0.0001) compared to empty vector PANC1 cells analyzed by MTS assay. (D) Increased gene expression of Ki-67 in R848 stimulated TLR7 + and TLR8 + PANC1 ( * P<0.0001 and ** P<0.0005) cells compared to empty vector PANC1 cells. Data of three independent experiments are shown with standard deviation. Untreated PANC1 cells were standardized to baseline. The relative gene expression is expressed as 2 −ΔΔCq .

Article Snippet: Blots were probed with antibodies to TLR7 (ProSci), TLR8 (ProSci), β-actin (Santa Cruz Biotechnology) and COX-2 (Santa Cruz Biotechnology and Novus Biologicals LLC, Littleton, CO, USA).

Techniques: Expressing, Injection, Plasmid Preparation, MTS Assay, Gene Expression, Standard Deviation

Journal: International Journal of Oncology

Article Title: TLR7 and TLR8 expression increases tumor cell proliferation and promotes chemoresistance in human pancreatic cancer

doi: 10.3892/ijo.2015.3069

Figure Lengend Snippet: NF-κB and COX-2 gene expression in response to R848 stimulation of TLR7 + and TLR8 + PANC1 cells. (A and B) Stimulation of TLR7 + and TLR8 + PANC1 cells with R848 resulted in significantly increased gene expression levels of NF-κB ( * P<0.0001) 6 h post stimulation. (C and D) Significantly escalated COX-2 gene expression levels 6–72 h after stimulation with maximum expression for TLR7 + PANC1 cells at 12 h ( ** P<0.005, *** P<0.05) and for TLR8 + PANC1 cells at 24 h ( * P<0.0001, ** P<0.005, *** P<0.05). Data of three independent experiments are shown with standard deviation. Untreated PANC1 cells were standardized to baseline. The relative gene expression is expressed as 2 −ΔΔCq .

Article Snippet: Blots were probed with antibodies to TLR7 (ProSci), TLR8 (ProSci), β-actin (Santa Cruz Biotechnology) and COX-2 (Santa Cruz Biotechnology and Novus Biologicals LLC, Littleton, CO, USA).

Techniques: Gene Expression, Expressing, Standard Deviation

Journal: International Journal of Oncology

Article Title: TLR7 and TLR8 expression increases tumor cell proliferation and promotes chemoresistance in human pancreatic cancer

doi: 10.3892/ijo.2015.3069

Figure Lengend Snippet: Effect of 5-fluorouracil on TLR7 + and TLR8 + PANC1 cells. (A) LD 50 concentration of 5-fluorouracil (5-FU) for empty vector PANC1 cells was found at a concentration of 500 μmol/l by MTS assay. (B) Decreased sensitivity of TLR7 + PANC1 cells ( *** P<0.05) and TLR8 + PANC1 cells ( * P<0.0001) to different concentrations of 5-FU compared to empty vector PANC1 cells analyzed by MTS assay. Untreated PANC1 cells were standardized to baseline. (C) Significantly reduced chemosensitivity of R848 and 5-FU (500 μmol/l) treated TLR7 + PANC1 cells and TLR8 + PANC1 cells compared to empty vector PANC1 cells ( * P<0.0001) found by MTS assay. Untreated PANC1 cells were standardized to baseline.

Article Snippet: Blots were probed with antibodies to TLR7 (ProSci), TLR8 (ProSci), β-actin (Santa Cruz Biotechnology) and COX-2 (Santa Cruz Biotechnology and Novus Biologicals LLC, Littleton, CO, USA).

Techniques: Concentration Assay, Plasmid Preparation, MTS Assay