Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: TLR3 activation augments matrix metalloproteinase production through reactive nitrogen species generation in human lung fibroblasts.

doi: 10.4049/jimmunol.1302919

Figure Lengend Snippet: FIGURE 9. Responsiveness of NHLFs and DHLF-As to poly(I:C). NHLFs (normal) (n = 3) and DHLF-As (asthma) (n = 4) were incubated with 30 mg/ml poly(I:C) or vehicle for 48 h. MMP-9 (A and B), -2 (A and C) and -1 (D) were analyzed by zymography. Fold increase was calculated by dividing the band intensity of MMPs in the poly(I:C)-treated group by that in the vehicle-treated group. The fold increases in the MMPs from NHLFs are compared with those from DHLF-As (B–D). Expression of TLR3 (E) and iNOS (F) in the cells was analyzed by immunoblotting. *p , 0.05 versus NHLFs. M, markers of MMPs.

Article Snippet: The cells were incubated with mouse monoclonal anti-TLR3 Ab (1:100 dilution; Imgenex) or mouse IgG as a negative control for detection of TLR3.

Techniques: Incubation, Zymography, Expressing, Western Blot

Journal: Journal of Virology

Article Title: Toll-Like Receptor Expression and Induction of Type I and Type III Interferons in Primary Airway Epithelial Cells

doi: 10.1128/jvi.01956-12

Figure Lengend Snippet: Figure 8. TLR3-poly I:C interaction mediates IFNλ production by AECs. (A) IFNλ 466

Article Snippet: Mouse TLR3 was detected with PE-conjugated anti-mouse 119 TLR3 (IgG1,κ) and its respective isotype control (both from Imgenex).

Techniques:

Journal: The Journal of Experimental Medicine

Article Title: Severe influenza pneumonitis in children with inherited TLR3 deficiency

doi: 10.1084/jem.20181621

Figure Lengend Snippet: Heterozygous TLR3 mutations in three unrelated IAV-ARDS patients. (A) Family pedigrees with TLR3 allele segregation. The black symbols indicate patients, and the bold vertical lines indicate healthy carriers of the mutant TLR3 allele. Healthy TLR3 WT relatives are indicated by white symbols. (B) Multiple sequence alignment across 19 vertebrate species. Both residues mutated in P1, P2, and P3 were highly conserved. (C) Predicted three-dimensional structure of the human TLR3 protein ectodomain. The residues affected by the two missense mutations found in IAV-ARDS patients are highlighted in magenta (P554S and P680L). The L360P mutation was previously identified in a patient with HSE (highlighted in violet). (D) Schematic diagram of the structure of the human TLR3 gene and protein, featuring the leader sequence (L), leucine-rich repeats (LRRs) of the ectodomain, transmembrane domain (TM), linker region (LR), and Toll/IL-1 receptor (TIR) domain. Roman numerals indicated the coding exons. Previously reported experimentally validated deleterious mutations found in HSE patients are shown in blue. The mutations found in the three children with severe influenza are shown in red (including the P554S mutation found in three previously reported and one unreported HSE patient).

Article Snippet: Equal amounts of protein from each sample were subjected to immunoprecipitation with a goat anti-human TLR3 antibody directed against the human TLR3 ectodomain (R&D Systems).

Techniques: Mutagenesis, Sequencing

Journal: The Journal of Experimental Medicine

Article Title: Severe influenza pneumonitis in children with inherited TLR3 deficiency

doi: 10.1084/jem.20181621

Figure Lengend Snippet: Expression and function of the P680L mutant TLR3 allele. (A) RT-qPCR for IFNB and IFNL1 mRNA expression without stimulation (NS) or after 2 and 4 h of stimulation with 25 µg/ml poly(I:C) in P2.1 cells not transfected (NT) or stably transfected with empty vector, HA-tagged TLR3 WT, P554S, or P680L. VSV M51R, at a MOI of 1, was used as a positive stimulus for IFN induction via the TLR3-independent pathways. Mean values ± SD were calculated from two independent experiments, with biological duplicates in each experiment. (B) TLR3 mRNA levels were determined by RT-qPCR in P2.1 TLR3-deficient fibrosarcoma cells with or without transfection with empty vector, HA-tagged TLR3 WT, P554S, or P680L. Mean values ± SD were calculated from two independent experiments, with biological duplicates in each experiment. (C) TLR3 was detected on immunoblots. P2.1 cells not transfected or stably transfected with empty vector, HA-tagged TLR3 WT, or P680L were subjected to immunoprecipitation (IP) with anti-TLR3 antibody, and the immunoprecipitated protein was then immunoblotted (IB) with C-ter (C) HA antibody or N-ter (N) TLR3 antibody. GAPDH was used as a loading control for immunoblotting. Reproducible result from six independent experiments is shown. (D–F) Immunofluorescence imaging of P2.1 cells stably expressing HA-tagged WT or P680L. Intracellular distribution was assessed by colocalization (Coloc) with a subcellular marker: anti-PDI antibody for the ER, anti-EEA1 antibody for early endosomes, and anti-LAMP1 antibody for lysosomes. Cells were let un-treated (E) or incubated with 25 µg/ml poly(I:C) for 30 min (F). The images were analyzed with Imaris Coloc software, and plots were generated. About 200 cells were used for each analysis. Mean values ± SD were calculated from two independent experiments. *, P < 0.05; ****, P < 0.0001.

Article Snippet: Equal amounts of protein from each sample were subjected to immunoprecipitation with a goat anti-human TLR3 antibody directed against the human TLR3 ectodomain (R&D Systems).

Techniques: Expressing, Mutagenesis, Quantitative RT-PCR, Transfection, Stable Transfection, Plasmid Preparation, Western Blot, Immunoprecipitation, Control, Immunofluorescence, Imaging, Marker, Incubation, Software, Generated

Journal: The Journal of Experimental Medicine

Article Title: Severe influenza pneumonitis in children with inherited TLR3 deficiency

doi: 10.1084/jem.20181621

Figure Lengend Snippet: Impaired poly(I:C) responses in SV40-fibroblasts from patients heterozygous for TLR3 P554S or P680L, and rescue by WT TLR3. (A–D) Production of IFN-β, IFN-λ (A and B), and IL-6 (C and D) in SV40-fibroblasts from three healthy controls (CTL1/2/3), P2 (A and C), P3 (B and D), a TLR3 P554S/WT HSE patient, and a TLR3 −/− HSE patient, 24 h after stimulation with 1, 5, or 25 µg/ml poly(I:C), or with 25 µg/ml poly(I:C) in the presence of lipofectamine (poly[I:C]+L; A and B), lipofectamine alone (L; A and B), or IL-1β (C and D), as assessed by ELISA. (E) IFNB , IFNL1 , and IL6 mRNA levels in SV40-fibroblasts from two CTLs, P3, P554S/WT, and TLR3 −/− patients, not stimulated (NS), or stimulated for 2 and 4 h with 25 µg/ml poly(I:C), or infected with VSV M51R at a MOI of 1 for 16 h. GUS was included for normalization. (F–I) Complementation of the impaired poly(I:C) response by introducing WT TLR3 into the patient’s fibroblasts. (F) IFNB , IFNL1 mRNA levels in SV40-fibroblasts from a healthy control (CTL) and P3, without plasmid transfection (NT) or after transfection with luciferase (Luc), Flag-tagged WT or P680L TLR3 , and in fibroblasts from a TLR3 −/− patient, not stimulated (NS), or stimulated for 2 h with 25 µg/ml poly(I:C), or infected with VSV M51R at a MOI of 1 for 16 h. GUS was included for normalization. (G) Production of IFN-λ, in the absence of stimulation, after 24 h of stimulation with 1, 5, or 25 µg/ml poly(I:C), and after stimulation with 25 µg/ml poly(I:C) in the presence of lipofectamine, or lipofectamine alone, as assessed by ELISA, in SV40-fibroblasts from a CTL and P3, without plasmid transfection or after transfection with Luc, Flag-tagged WT or P680L TLR3 , and in fibroblasts from a TLR3 −/− patient. (H) TLR3 mRNA levels were assessed by RT-qPCR. (I) TLR3 was detected on immunoblots following IP. Mean values ± SD were calculated from four (A–D and H) or two (E and F) independent experiments, with technical duplicates in each experiment. (G) Results from a single experiment with biological duplicates, representing three independent experiments. (I) Reproducible results from three independent experiments.

Article Snippet: Equal amounts of protein from each sample were subjected to immunoprecipitation with a goat anti-human TLR3 antibody directed against the human TLR3 ectodomain (R&D Systems).

Techniques: Enzyme-linked Immunosorbent Assay, Infection, Control, Plasmid Preparation, Transfection, Luciferase, Quantitative RT-PCR, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: Severe influenza pneumonitis in children with inherited TLR3 deficiency

doi: 10.1084/jem.20181621

Figure Lengend Snippet: Normal IFN response to poly(I:C) and viruses in TLR3-mutated PBMCs. (A) PBMCs from four CTLs, P3, P3’s parents, and a TLR3 −/− patient were infected with various types of viruses: double-stranded DNA (dsDNA; HSV-1) and single-stranded RNA (ssRNA − ; IAV strain pH1N1, VSV, Sendai virus, mumps virus, measles virus, HPIV3) viruses. The levels of IFN-α, -β, and -λ and IL-6 were measured by ELISA 24 h after infection. (B) The induction of IFNL1, IFNB, MX1, OAS1 , and ISG15 mRNA was assessed by RT-qPCR in PBMCs from four CTLs, P3, P3’s parents, and a TLR3 −/− patient. RNA was isolated from these cells after 8 h of poly(I:C) stimulation and 24 h of IAV pH1N1 and HSV-1 infection. Mean values ± SD were calculated from biological duplicates from two independent experiments.

Article Snippet: Equal amounts of protein from each sample were subjected to immunoprecipitation with a goat anti-human TLR3 antibody directed against the human TLR3 ectodomain (R&D Systems).

Techniques: Infection, Virus, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Isolation

Journal: The Journal of Experimental Medicine

Article Title: Severe influenza pneumonitis in children with inherited TLR3 deficiency

doi: 10.1084/jem.20181621

Figure Lengend Snippet: Enhanced susceptibility to IAV in TLR3-mutated fibroblasts, and rescue by WT TLR3. (A) IAV replication, quantified by plaque assays, in SV40-fibroblasts from three CTLs, P3, P554S/WT, TLR3 −/− , IRF7 −/− , and STAT1 −/− patients, 1, 8, 12, 24, and 36 h after infection at a MOI of 10. Cells were untreated or subjected to pretreatment with IFN-α2b, IFN-β, or IFN-λ for 16 h before infection. The data shown are representative of three independent experiments, with biological duplicates in each experiment. (B) Production of IFN-β and IFN-λ in the absence of infection or after 24 h of infection with IAV at a MOI of 1, in SV40-fibroblasts from seven CTLs, P2, P3, a TLR3 P554S/WT HSE patient, TLR3 −/− , IRF7 −/− , and NF-κB essential modulator (NEMO)–deficient patients, as assessed by ELISA. (C) Production of IFN-β and IFN-λ in the absence of infection or after 24 h of infection with IAV at a MOI of 5 or 10, in SV40-fibroblasts from a CTL and P3, without plasmid transfection or after transfection with Luc, Flag-tagged WT TLR3 , and in fibroblasts from a TLR3 −/− patient. (D) IAV replication, quantified by plaque assays, in SV40-fibroblasts from two CTLs and P3, without plasmid transfection or after transfection with Luc, Flag-tagged WT TLR3 , and in fibroblasts from a TLR3 −/− patient, 1, 8, 12, 24, and 36 h after infection at a MOI of 5. Mean values ± SD from five (B) or three (C and D) independent experiments are shown. Biological duplicates were tested in each experiment. **, P < 0.01; ***, P < 0.001.

Article Snippet: Equal amounts of protein from each sample were subjected to immunoprecipitation with a goat anti-human TLR3 antibody directed against the human TLR3 ectodomain (R&D Systems).

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Transfection

Journal: The Journal of Experimental Medicine

Article Title: Severe influenza pneumonitis in children with inherited TLR3 deficiency

doi: 10.1084/jem.20181621

Figure Lengend Snippet: Enhanced susceptibility to IAV in TLR3-mutated iPSC-derived lung epithelial cells. Lung epithelial cells were derived from the ES cells of a healthy control (CTL-RUES2, shown as black filled circles in the figure), iPSCs from two healthy controls (Sendai virus–reprogrammed iPSC CTL-SViPS, and mRNA-reprogrammed iPSC CTL-mRNA, shown as dark or light blue triangles, respectively), P3 (three iPSC clones from the same patient, shown with pink squares, yellow triangles, or red circles, respectively), TLR3 −/− , IRF7 −/− , STAT1 −/− , and IL10RB −/− patients. The cells were untreated or subjected to 16 h of pretreatment with IFN-α2b or IFN-λ1, then infected with IAV at a MOI of 1 or 10 for 24 h. The cells were immunostained for influenza NP (green) and Nkx2.1 (red), and their nuclei were stained with DAPI (blue). (A) The percentage of Nkx2.1-positive cells was determined for the DAPI-positive cells. (B) Representative images of CTL and patient PECs, showing the immunostaining of NP, Nkx2.1, and DAPI, 24 h after infection with IAV. hES, human embryonic stem. (C) The percentage of influenza NP-positive cells was then determined for the Nkx2.1-positive cells. We analyzed ∼60,000 Nkx2.1 cells per cell line. Without IFN pretreatment, higher proportions of PECs derived from the iPSCs of TLR3 P680L/WT, TLR3 −/− , IRF7 −/− , STAT1 −/− , and IL10RB −/− patients were positive for IAV NP, than of PECs from CTL-RUES2, iPSC CTL-SViPS, and iPSC CTL-mRNA. The data shown represent the mean values ± SD from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Equal amounts of protein from each sample were subjected to immunoprecipitation with a goat anti-human TLR3 antibody directed against the human TLR3 ectodomain (R&D Systems).

Techniques: Derivative Assay, Control, Virus, Clone Assay, Infection, Staining, Immunostaining

Journal: Journal of Biological Chemistry

Article Title: Recognition of Double-stranded RNA by Human Toll-like Receptor 3 and Downstream Receptor Signaling Requires Multimerization and an Acidic pH

doi: 10.1074/jbc.m507163200

Figure Lengend Snippet: FIGURE 1. Cysteine residues and glycosylation of TLR3 leucine-rich repeats are necessary for ligand-induced signaling. A, TLR3 expression in HEK293 and HEK293T by Western blot. Whereas HEK293 cells express TLR3, HEK293T cells did not, except when transfected with the TLR3pMET7 wild type construct (500 ng). B and C, Western blot analysis of wild type TLR3 and mutants. HEK293T cells were transfected with 500 ng of TLR3pMET7 wild type or TLR3pMET7 cysteine (B) or glycosylation (C) mutants. 24 h post-transfection, cells were lysed, and TLR3 expression was determined by Western blotting under reduced and nonreduced conditions using an anti-TLR3 antibody. D, reporter assay in HEK293T cells. 250 ng ofwildtypeTLR3ormutantswereco-transfectedwith500ngofNF-Bluciferase.LuciferaseactivityofTLR3mutantsweremeasuredinresponsetostimulationwith5g/mlpoly(I-C). E, position of the described mutants within the first 10 leucine-rich repeat motifs of TLR3. Leucine-rich repeats are shown aligned with the consensus sequence of the -sheet and -helix (marked above the alignment). Mutated residues are boxed, and the exact residue number is indicated by an arrow and the position on the sequence. From this simple alignment, it can be seen that the paired cysteine residues (Cys95 and Cys122; Cys216 and Cys242) lie in adjacent helical regions. The two N-glycosylation sites (Asn196 and Asn247) are positioned in an - turn structure in repeats slightly longer than the average, suggesting that they are highly accessible.

Article Snippet: After incubation with anti-TLR3 antibody (clone 40C1285; Imgenex, Sorrento Valley, CA), reactive proteins were detected with peroxidaseconjugated anti-mouse secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., Soham, Cambridgeshire, UK) and ECL (Amersham Biosciences).

Techniques: Glycoproteomics, Expressing, Western Blot, Transfection, Construct, Reporter Assay, Sequencing, Residue

Journal: Journal of Biological Chemistry

Article Title: Recognition of Double-stranded RNA by Human Toll-like Receptor 3 and Downstream Receptor Signaling Requires Multimerization and an Acidic pH

doi: 10.1074/jbc.m507163200

Figure Lengend Snippet: FIGURE 2. Use of loss of function TLR3 mutants suggests that TLR3 multimerization was required for signaling. An NF-B reporter competition assay with increasing amounts of TLR3 cysteine (A and C) or glycosylation (B and D) mutants versus endogenous wild type TLR3 in HEK293 cells (A and B) or versus 500 ng of wild type TLR3pMET7 transfected in HEK293T cells (C and D) was performed. 100, 250, and 500 ng, respectively, of TLR3 mutants were co-transfected with 500 ng of NF-B luciferase. 24 h post-transfection, the luciferase activity of transfectants was measured in response to stimulation with 5 g/ml poly(I-C).

Article Snippet: After incubation with anti-TLR3 antibody (clone 40C1285; Imgenex, Sorrento Valley, CA), reactive proteins were detected with peroxidaseconjugated anti-mouse secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., Soham, Cambridgeshire, UK) and ECL (Amersham Biosciences).

Techniques: Competitive Binding Assay, Glycoproteomics, Transfection, Luciferase, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Recognition of Double-stranded RNA by Human Toll-like Receptor 3 and Downstream Receptor Signaling Requires Multimerization and an Acidic pH

doi: 10.1074/jbc.m507163200

Figure Lengend Snippet: FIGURE 3. Construction and expression of the humanTLR3-CD32chimera.A,achimericrecom- binant molecule between human TLR3 and human CD32a was created using the whole extra- cellular amino-terminal portion of hTLR3 (EC- TLR3) and the whole intracellular carboxyl-termi- nal part of CD32a (C term-CD32), including its transmembrane domain and conserved ITAM. EC- TLR3 from nucleotide 102 to 2212 (GenBankTM

Article Snippet: After incubation with anti-TLR3 antibody (clone 40C1285; Imgenex, Sorrento Valley, CA), reactive proteins were detected with peroxidaseconjugated anti-mouse secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., Soham, Cambridgeshire, UK) and ECL (Amersham Biosciences).

Techniques: Expressing

Journal: Journal of Biological Chemistry

Article Title: Recognition of Double-stranded RNA by Human Toll-like Receptor 3 and Downstream Receptor Signaling Requires Multimerization and an Acidic pH

doi: 10.1074/jbc.m507163200

Figure Lengend Snippet: FIGURE 4. Use of anti-TLR3 antibodies shows the requirement of cross-linking for TLR3-CD32 signalization. Ca2 flux was induced by anti-CD32 antibody but only upon cross-linking with secondary reagent on myelomonocytic cell line U937 (A). Using the TLR3-CD32 chimera stably transfected U937, clone 2H4, a goat (C) or mouse (D) anti-TLR3 antibodies (0.5 and 2.5 g/ml, respectively) were inactive alone but triggered Ca2 flux upon cross-linking with their respective conjugate (10 g/ml). No signal was observed on empty vector-transfected cells even after cross-linking (B). Anti-TLR3 cross-linked response desensitized a secondary response to 25 g of poly(I-C) (C and D) and vice versa (E and F).

Article Snippet: After incubation with anti-TLR3 antibody (clone 40C1285; Imgenex, Sorrento Valley, CA), reactive proteins were detected with peroxidaseconjugated anti-mouse secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., Soham, Cambridgeshire, UK) and ECL (Amersham Biosciences).

Techniques: Stable Transfection, Transfection, Plasmid Preparation

Journal: Journal of Biological Chemistry

Article Title: Recognition of Double-stranded RNA by Human Toll-like Receptor 3 and Downstream Receptor Signaling Requires Multimerization and an Acidic pH

doi: 10.1074/jbc.m507163200

Figure Lengend Snippet: FIGURE 5. Poly(I-C) induces Ca2 flux on U937 cells stably transfected with the TLR3-CD32 chimera. Calcium flux is induced by poly(I-C) (25 g/ml) on the TLR3-CD32 chimera stably transfected U937, clone 2H4 (A), but not on the TLR2-CD32-transfected cells (B). However, cross-linking with a mouse anti-TLR2 plus anti-mouse conjugate induced a flux. A first injection of poly(I-C) (10 g) desensitized a second injection of poly(I-C) (25 g) (C), and 10 g of poly(A-U) induced a Ca2 flux that desensitized the response to poly(I-C) (25 g) (D) and vice versa (not shown). The dose response to poly(I-C) on the stable clone 2H4 (E) compared with the IP-10/CXCL10 secretion on PBMC (F) shows that the Ca2 flux sensitivity through the chimera was stronger than the ex vivo endogenous TLR3 response in immune cells.

Article Snippet: After incubation with anti-TLR3 antibody (clone 40C1285; Imgenex, Sorrento Valley, CA), reactive proteins were detected with peroxidaseconjugated anti-mouse secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., Soham, Cambridgeshire, UK) and ECL (Amersham Biosciences).

Techniques: Stable Transfection, Transfection, Injection, Ex Vivo

Journal: Journal of Biological Chemistry

Article Title: Recognition of Double-stranded RNA by Human Toll-like Receptor 3 and Downstream Receptor Signaling Requires Multimerization and an Acidic pH

doi: 10.1074/jbc.m507163200

Figure Lengend Snippet: FIGURE 6. The intensity of the Ca2 response was proportional to the size of the synthetic dsRNA. Poly(I-C)s of defined size were tested on the TLR3-CD32 chimera stably transfected U937, clone 2H4. The concentration of small fragments ((I-C)15, (I-C)30, and (I-C)40) necessary to induce Ca2 flux is higher compared with high molecular weight poly(I-C) (A) (ND, not done). 10 g/ml small size poly(I-C) synthetic fragments (I-C)15 (B) and (I-C)40 (C) of 15 and 40 base pairs, respectively, induced a partial Ca2 flux, as seen by residual secondary response to 25 g of poly(I-C). In contrast, 10 g of 100–200-bp hydrolysis-fragmented poly(I-C) induced a complete Ca2 response (D).

Article Snippet: After incubation with anti-TLR3 antibody (clone 40C1285; Imgenex, Sorrento Valley, CA), reactive proteins were detected with peroxidaseconjugated anti-mouse secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., Soham, Cambridgeshire, UK) and ECL (Amersham Biosciences).

Techniques: Stable Transfection, Transfection, Concentration Assay, High Molecular Weight

Journal: Journal of Biological Chemistry

Article Title: Recognition of Double-stranded RNA by Human Toll-like Receptor 3 and Downstream Receptor Signaling Requires Multimerization and an Acidic pH

doi: 10.1074/jbc.m507163200

Figure Lengend Snippet: FIGURE 8. Effect of pH on poly(I-C)-mediated TLR3 activation. Poly(I-C)-mediated TLR3 Ca2 flux response is observed at acidic pH (pH 5.7–6.7) and blocked at neutral to basic pH (from pH 7), whereas the antibody-mediated response was unaffected (exam- ple in A and pH curve in B). Small fragments (10 g/ml) are even more sensitive to pH variation than poly(I-C) and poly(A-U) (C). As an example, the activity of poly(I-C)15 is optimalatpH5.7,desensitizingasecondaryresponsetohighmolecularweightpoly(I-C), and decreases rapidly at pH above 6.25, whereas that of high molecular weight poly(I-C) remains optimal up to pH 6.6 (D)

Article Snippet: After incubation with anti-TLR3 antibody (clone 40C1285; Imgenex, Sorrento Valley, CA), reactive proteins were detected with peroxidaseconjugated anti-mouse secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., Soham, Cambridgeshire, UK) and ECL (Amersham Biosciences).

Techniques: Activation Assay, Activity Assay, High Molecular Weight

Journal: Cells

Article Title: Distinct Effects of Respiratory Viral Infection Models on miR-149-5p, IL-6 and p63 Expression in BEAS-2B and A549 Epithelial Cells

doi: 10.3390/cells13110919

Figure Lengend Snippet: TLR3 expression in airway epithelial cells at baseline and after incubation with poly (I:C). BEAS-2B or A549 cells were cultured in a 24-well plate at 1 × 10 5 cells per well in a BEGM or F-12K medium (FBS, 10%) for 24 h. BEAS-2B or A549 cells were incubated in a BEBM or F-12K medium containing ITS+1 (1%) with poly (I:C) (0.5 μg/mL) for 24 or 48 h. ( a ) TLR3 mRNA expression at baseline in BEAS-2B and A549 cells, n = 3. The Ct value of TLR3 was normalised to that of 18S rRNA (ΔCt). Data are presented relative to the BEAS-2B cells (ΔΔCt) as mean ± SEM. ( b ) Representative immunoblot and ( c ) densitometry analysis of TLR3 (125 kDa) at baseline in BEAS-2B cells and A549 cells, n = 3. GAPDH (37 kDa) was used as a loading control. Data are presented as mean ± SEM. Values were normalised to GAPDH as a loading control. ( d ) TLR3 mRNA expression in BEAS-2B cells challenged with poly (I:C). The cycle threshold (Ct) value of TLR3 was normalised to that of 18S rRNA (ΔCt). Data are presented relative to the control at baseline (ΔΔCt), n = 3. ( e ) Representative immunoblot and ( f ) densitometry analysis of TLR3 in BEAS-2B cells incubated with poly (I:C), n = 5. ( g ) TLR3 mRNA expression in A549 cells challenged with poly (I:C), n = 3. ( h ) Representative immunoblot of TLR3 in A549 cells incubated with poly (I:C), n = 3. Data are presented as mean ± SEM. * p ≤ 0.05, compared with the control group, using unpaired t -test or one-way analysis of variance with Bonferroni post-test.

Article Snippet: The following antibodies were used for immunoblotting: purified anti-p63 (ΔN) antibody (Cell Signaling Technology, 67825, Danvers, MA, USA), anti-TLR3 antibody (R&D systems, Bio-techne, AF1487, Abingdon, UK), anti-TLR2 antibody (R&D systems, Bio-techne, AF2616), anti-GAPDH antibody (Abcam, ab9483, Cambridge, UK), donkey anti-goat IgG (HRP, Abcam, ab205723) and goat anti-rabbit IgG (HRP, Abcam, ab6721).

Techniques: Expressing, Incubation, Cell Culture, Western Blot, Control