tlr2 cdna  (Addgene inc)

Journal: Molecular oral microbiology

Article Title: Porphyromonas gingivalis RagB is a pro-inflammatory STAT4 agonist

doi: 10.1111/omi.12089

Figure Lengend Snippet: Primary human monocytes were untreated or exposed to combinations of RagB (1 μg/ml); the TLR2 agonist, Pam3CSK (1 μg/ml), anti-TLR2 (1 μg/ml, 1 hr pre- treatment) and control (IgA2, 1μg/ml, 1 hr pre-treatment) antibodies. In an alternative strategy, TLR2 gene silencing was performed, as described in the text.

Article Snippet: Materials P. gingivalis W83 was purchased from the American Type Culture Collection (Manassas, VA); Gifu Anaerobe Medium (GAM) from Nissui Pharmaceutical (Tokyo, Japan); Δ ragB and pTCBex2: ragB W83-carrying complemented P. gingivalis strains were kindly provided by Dr. Fuminobu Yoshimura (Aichi-Gakuin University, Nagoya, Japan) and have been described previously ( Nagano et al. , 2007 ) and; lymphocyte separation medium (LSM; Fischer Scientific, Pittsburgh, PA); CD14 microbeads from Miltenyi Biotec (Auburn, CA); Escherichia coli TOP10, TOPO expression kits and 5-Prime RNA kits from 5-Prime (Gaithersburg, MD); cytokine ELISAs came from R&D systems (Minneapolis, MN) and eBioscience (San Diego, CA); Human Toll-Like Receptor (TLR) Signaling Pathway RT 2 Profiler PCR array and RT 2 SYBR Green ROX qPCR Mastermix came from Quiagen (Valencia, CA); whole genome microarrays were from Affymetrix (Santa Clara, CA); siRNA (TLR4, sc-40260: TLR2, sc-40256: p52, sc-29409: p65, sc-29410; STAT4, sc-36568: control, sc-37007) from Santa Cruz Biotechnology (Santa Cruz, CA); ultrapure E. coli K12 LPS, anti-TLR2 and -4 antibodies (clones B4H2 and W7C11, respectively), qScript cDNA SuperMix from Quanta BioSciences Inc. (Gaithersburg, MD); THP1-XBlue NF-kB/AP-1 SEAP Reporter cells and TLR-transfected HEK293 cells came from InvivoGen (Santa Clara, CA); the STAT4 inhibitor, lysofylline (1-[(5 R )-5-Hydroxyhexyl]-3,7-dimethyl-3,7-dihydro-1 H -purine-2,6-dione; LSF), was from Cayman Chemical Co (Ann Arbor, MI); the p65 (Ser276) inhibitory peptide set came from Novus Biologicals (Littleton, CO); while LB agar, kanamycin, arabinose, imidazole, RPMI 1640, fetal bovine serum (FBS), penicillin G, streptomycin, proteinase K, polymyxin B and agarose came from Sigma-Aldrich (St. Louis, MO).

Techniques:

Journal: Molecular oral microbiology

Article Title: Porphyromonas gingivalis RagB is a pro-inflammatory STAT4 agonist

doi: 10.1111/omi.12089

Figure Lengend Snippet: Primary human monocytes were untreated or exposed to combinations of RagB (1 μg/ml); the TLR2 agonist, E. coli LPS (1μg/ml), anti-TLR4 (1μg/ml), 1 hr pre- treatment) and control (IgG1, 1μg/ml), 1 hr pre- treatment) antibodies. In an alternative strategy, TLR4 gene silencing was performed, as described in the text.

Article Snippet: Materials P. gingivalis W83 was purchased from the American Type Culture Collection (Manassas, VA); Gifu Anaerobe Medium (GAM) from Nissui Pharmaceutical (Tokyo, Japan); Δ ragB and pTCBex2: ragB W83-carrying complemented P. gingivalis strains were kindly provided by Dr. Fuminobu Yoshimura (Aichi-Gakuin University, Nagoya, Japan) and have been described previously ( Nagano et al. , 2007 ) and; lymphocyte separation medium (LSM; Fischer Scientific, Pittsburgh, PA); CD14 microbeads from Miltenyi Biotec (Auburn, CA); Escherichia coli TOP10, TOPO expression kits and 5-Prime RNA kits from 5-Prime (Gaithersburg, MD); cytokine ELISAs came from R&D systems (Minneapolis, MN) and eBioscience (San Diego, CA); Human Toll-Like Receptor (TLR) Signaling Pathway RT 2 Profiler PCR array and RT 2 SYBR Green ROX qPCR Mastermix came from Quiagen (Valencia, CA); whole genome microarrays were from Affymetrix (Santa Clara, CA); siRNA (TLR4, sc-40260: TLR2, sc-40256: p52, sc-29409: p65, sc-29410; STAT4, sc-36568: control, sc-37007) from Santa Cruz Biotechnology (Santa Cruz, CA); ultrapure E. coli K12 LPS, anti-TLR2 and -4 antibodies (clones B4H2 and W7C11, respectively), qScript cDNA SuperMix from Quanta BioSciences Inc. (Gaithersburg, MD); THP1-XBlue NF-kB/AP-1 SEAP Reporter cells and TLR-transfected HEK293 cells came from InvivoGen (Santa Clara, CA); the STAT4 inhibitor, lysofylline (1-[(5 R )-5-Hydroxyhexyl]-3,7-dimethyl-3,7-dihydro-1 H -purine-2,6-dione; LSF), was from Cayman Chemical Co (Ann Arbor, MI); the p65 (Ser276) inhibitory peptide set came from Novus Biologicals (Littleton, CO); while LB agar, kanamycin, arabinose, imidazole, RPMI 1640, fetal bovine serum (FBS), penicillin G, streptomycin, proteinase K, polymyxin B and agarose came from Sigma-Aldrich (St. Louis, MO).

Techniques:

Journal: bioRxiv

Article Title: Phosphatidylserine clustering by membrane receptors triggers LC3-associated phagocytosis

doi: 10.1101/2023.09.06.556449

Figure Lengend Snippet: ( a ) Principal component analysis of lipid content from phagosomes containing Pam3csk4-beads (Pam3-phag, blue) relative to phagosomes containing uncoupled-beads (control-phag, grey) isolated from immortalized bone marrow derived macrophages (iBMDM) in a representative experiment, n=4. ( b ) Fold change of MS/MS values of different lipid species in phagosomes containing Pam3-phag relative to control-phag isolated from iBMDM. Lipid species determined by lipidomics were aggregated per lipid class and each dot represents the cumulative value in n=4 independent experiments. PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; Cer, Ceramide; DG, diacylglycerol; TG, triacylglycerol. ( c , d ) RAW264.7 cells stably expressing the PS-probes Venus-LACT-C2 or Venus-FVIII-C2 were fed BSA-beads, BSA-beads coupled with anti-BSA antibody (IgG-BSA-beads), or Pam3csk4-beads (Pam3-beads) for 30min and enrichment of PS-probe to phagosome membranes relative to cytosolic signal was determined by immunofluorescence. Each dot represents a phagosome (n>10) in a representative experiment, n=2. ( e ) Cumulative MS/MS peak area of PC and PS of Pam3-phag from wild-type (WT) and TLR2-KO iBMDM determined by lipidomic analysis. Replicates in a representative experiment, n=2. ( f ) Scheme of mouse TLR2 (Uniprot: Q9QUN7) depicted in a membrane. Transmembrane domain is highlighted in yellow; acidic and basic amino acid are colored in red and blue, respectively; and Toll-Interleukin receptor (TIR) domain is boxed in magenta. Numbers indicate amino acid position showcasing the basic patch ( 628 KRKPKK 6 ). ( g - j ) RAW264.7-sg-TLR2-KO cells stably expressing the PS-probe Venus-LACT-C2 ( g , h ) or Venus-LC3 ( i, j ) were transduced to express TLR2 full length or lacking the TIR domain (TLR2ΔTIR), in either wild-type (WT), K628E-R629D-K630E-K632E-K633E (ACID), P631H (corresponding to human SNP rs5743704), or P681H mutant TLR2. Transduction with empty vector (e.v.) served as a negative control. Cells were fed Pam3csk4-beads (30min, g, h; 1h, i, j) and Venus-LACT-C2 or Venus-LC3 translocation to phagosomes was determined by immunofluorescence. (g, i) Representative confocal images and (h, j) violin-plots of PS-probe enrichment at the phagosome membrane relative to cytosolic signal (n>20 phagosomes) in one representative experiment, n=2. ** P < 0.01, **** P < 0.001, *** P < 0.0001 by two-sided Student’s t test.

Article Snippet: TLR2 cDNA was a kind gift from Dr. Ruslan Medzhitov (Addgene#13083).

Techniques: Control, Isolation, Derivative Assay, Tandem Mass Spectroscopy, Stable Transfection, Expressing, Immunofluorescence, Membrane, Mutagenesis, Transduction, Plasmid Preparation, Negative Control, Translocation Assay

Journal: bioRxiv

Article Title: Phosphatidylserine clustering by membrane receptors triggers LC3-associated phagocytosis

doi: 10.1101/2023.09.06.556449

Figure Lengend Snippet: ( a ) PS-bead pull-down of TLR2 intracellular domain (TLR2-ID). Uncoupled-beads (Ctrl-beads), PS-beads, in combination with wild-type (WT), K628A-R629A-K630A-K632A-K633A (ALA), and K628E-R629D-K630E-K632E-K633E (ACID) versions were used as indicated, blots representative of n=4. ( b - f ) Glass-supported lipid bilayer resembling plasma membrane composition were incubated with TLR2-ID (b-c), cytosolic tails of mCD16 (d-f) or mTIM4 (d) as indicated, and top-Fluor-PS clustering was determined by immunofluorescence. (b, e) Representative images or (c, d, f) area of clustered PS quantified in different fields from representative experiments, n=2. (b-d) Anti-FLAG or streptavidin allows peptide manipulation and disrupts lipid clustering. **** P < 0.001 by two-sided Student’s t test.

Article Snippet: TLR2 cDNA was a kind gift from Dr. Ruslan Medzhitov (Addgene#13083).

Techniques: Clinical Proteomics, Membrane, Incubation, Immunofluorescence

Journal: bioRxiv

Article Title: Phosphatidylserine clustering by membrane receptors triggers LC3-associated phagocytosis

doi: 10.1101/2023.09.06.556449

Figure Lengend Snippet: ( a, b ) RAW264.7 cells were treated with ionomycin (10μM, 30min) combined with anti-phosphatidylserine (PS) antibody (1:50) or isotype control (anti-FLAG, 1:50) and fed Zymosan (30min). Representative confocal images (a) and violin-plots (b) of endogenous Rubicon enrichment at the phagosome membrane relative to cytosolic signal (n>40 phagosomes). ( c ) RAW264.7-sg-TLR2-KO cells were transduced to express full length TLR2 or TLR2 lacking the TIR domain (TLR2τιTIR), in either wild-type (WT), K628E-R629D-K630E-K632E-K633E (ACID), P631H (corresponding to human SNP rs5743704), or P681H mutant TLR2. Transduction with empty vector (e.v.) serves as a negative control. Cells were fed Pam3csk4-beads (30min) and Rubicon translocation was determined by immunofluorescence. Violin-plots showing the enrichment of Rubicon at the phagosome membrane relative to the cytosolic level (n>20 phagosomes). ( d, e ) RAW264.7-Rubicon-KO cells stably expressing the PS-probe Venus-FVIII-C2 and mCherry-Rubicon were fed Zymosan and analyzed by stochastic optical reconstruction microscopy (STORM). (d) Super-resolution image showing a representative membrane portion of a Zymosan-containing phagosome and (e) statistical index in super-resolution images assessing the proximity of PS-probe and Rubicon at phagosome membranes over time. Data are means ± SD of >6 biological replicates (phagosomes) in one representative experiment. ( f ) Lipid strip showing the lipid-binding specificity of full-length Rubicon recombinantly produced in insect cells, n=2. ( g ) In vitro binding competition assays of proteins in Pam3csk4-beads containing phagosomes isolated from RAW264.7-Rubicon-KO cells. Phagosomes were incubated with Lactadherin-FITC (Lact-FITC) and/or GST-Rubicon D1-644 as indicated. After anti-GST-Cy3 staining, phagosomes were quantitively analyzed by microscopy for green (Lactadherin binding) or red signal (GST-Rubicon Δ1-644 binding). Violin plots depict n>30 phagosomes. ** P <0.01, *** P <0.001, **** P <0.0001 by (b, g) two-sided Student’s t test or (c) ANOVA test (pairwise comparations Fisher’s LSD).

Article Snippet: TLR2 cDNA was a kind gift from Dr. Ruslan Medzhitov (Addgene#13083).

Techniques: Control, Membrane, Mutagenesis, Transduction, Plasmid Preparation, Negative Control, Translocation Assay, Immunofluorescence, Stable Transfection, Expressing, Microscopy, Stripping Membranes, Binding Assay, Produced, In Vitro, Isolation, Incubation, Staining

Journal: Science Advances

Article Title: The innate immune sensor Toll-like receptor 2 controls the senescence-associated secretory phenotype

doi: 10.1126/sciadv.aaw0254

Figure Lengend Snippet: ( A ) Schematic showing IMR90 ER:RAS cells treated with 4OHT undergo OIS. IMR90 ER:STOP cells serve as a control and retain proliferative capacity with 4OHT. ( B ) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of TLR family member expression in IMR90 ER:RAS and ER:STOP cells treated with 4OHT for 5 and 8 days. ( C ) Western blot of TLR2 expression in IMR90 ER:RAS and ER:STOP (ER:S) cells with up to 10 days of 4OHT treatment (top). The 5-bromo-2′-deoxyuridine (BrdU) incorporation in IMR90 ER:RAS cells treated with 4OHT for up to 8 days, as indicated. ( D ) RNA was extracted from snap-frozen liver samples from wild-type (WT) mice 6 days following hydrodynamic delivery of Nras G12V/D38A ( n = 5) and Nras G12V ( n = 4) transposons. tlr2 mRNA expression was measured using qRT-PCR. Scatter plots represents value per animal, with the horizontal line representing group means ± SEM. Statistical significance was calculated using Students two-tailed t test. ** P < 0.01. ( E ) Immunohistochemical staining for Nras and Tlr2 in consecutive liver sections from corresponding mice in (D) showing that oncogenic Nras G12V expressing, but not Nras G12V/D38A expressing, hepatocytes express Tlr2. Scale bars, 50 μm.

Article Snippet: TLR2 complementary DNA (cDNA) was amplified from the pcDNA3-TLR2-YFP plasmid (Addgene, 13016) using primers flanked with Xho I sites.

Techniques: Control, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Western Blot, BrdU Incorporation Assay, Two Tailed Test, Immunohistochemical staining, Staining

Journal: Science Advances

Article Title: The innate immune sensor Toll-like receptor 2 controls the senescence-associated secretory phenotype

doi: 10.1126/sciadv.aaw0254

Figure Lengend Snippet: ( A ) Immunofluorescence staining and high-content analysis for IL-1β expression in IMR90 ER:RAS cells treated with 4OHT for 8 days and repeatedly transfected with pooled siRNA targeting TLR1, TLR2, TLR6, and TLR10. Nontarget (NT)-pooled siRNA was used as control. Representative images are shown. Scale bars, 250 μm. DAPI, 4′,6-diamidino-2-phenylindole. ( B ) Western blot analysis against indicated antibodies in IMR90 ER:RAS cells treated with 4OHT for 8 days and repeatedly transfected with pooled and individual siRNA targeting TLR2 and TLR10. T2-P, siRNA TLR2 pool; T2-4, individual TLR2 siRNA; T10-P, siRNA TLR10 pool; T10-2, individual TLR10 siRNA. Nontarget pooled siRNA was used as control. Western blot against β-actin is shown as a loading control. ( C ) SASP factor regulation by qRT-PCR in ER:RAS cells treated with 4OHT for 8 days and repeatedly transfected with pooled siRNA targeting TLR2 and TLR10. Results are expressed as means ± SEM of three independent experiments. ( D ) Venn diagram showing the number of genes that are significantly induced by TLR2 and TLR10 during OIS in the transcriptome analysis (AmpliSeq) in IMR90 ER:RAS cells treated with 4OHT and transfected with pooled siRNA targeting TLR2 and TLR10 for 8 days (GSE127116). The intersection represents the number of genes regulated by both TLR2 and TLR10. This signature of 267 genes will be used for GSEA in additional senescence transcriptomes in and figs. S4 and S7. ( E ) Top regulated terms identified through of coregulated genes in (H) using DAVID gene ontology analysis. Chart bars represent Benjamin-adjusted P value of term enrichment. ( F ) Heat map of SASP factor expression obtained from the transcriptome analysis (AmpliSeq) in IMR90 ER:RAS cells following siRNA knockdown of TLR2 and TLR10 for 8 days of 4OHT treatment. ( G ) GSEA enrichment plot of RELA signature in TLR2 siRNA-transfected IMR90 ER:RAS 4OHT-induced cells. ( H ) IMR90 ER:RAS cells were transfected with indicated siRNA for 8 days with 4OHT. Western blot for phosphorylation and total levels of IKKa/β and p38 mitogen-activated protein kinase (MAPK) was performed. ( I ) IMR90 ER:RAS cells were treated with 4OHT and repeatedly transfected with indicated pooled siRNA and nontarget siRNA as control for 5 days. Western blots were conducted for phosphorylation of p65 and total p65 protein levels. All statistical significance was calculated using one-way analysis of variance (ANOVA). *** P < 0.001, ** P < 0.01, and * P < 0.05. ns, not significant.

Article Snippet: TLR2 complementary DNA (cDNA) was amplified from the pcDNA3-TLR2-YFP plasmid (Addgene, 13016) using primers flanked with Xho I sites.

Techniques: Immunofluorescence, Staining, High Content Screening, Expressing, Transfection, Control, Western Blot, Quantitative RT-PCR, Knockdown, Phospho-proteomics

Journal: Science Advances

Article Title: The innate immune sensor Toll-like receptor 2 controls the senescence-associated secretory phenotype

doi: 10.1126/sciadv.aaw0254

Figure Lengend Snippet: ( A ) IMR90 cells infected with TLR2 or H-Ras G12V expression vectors or empty vector (EV) control were seeded at low density and stained with crystal violet after 2 weeks. The staining was quantified to obtain relative cell content. Results are expressed as means ± SEM of three independent experiments. ( B ) SA-β-Gal staining was carried out on TLR2- and H-Ras G12V –expressing cells. Results are expressed as means (% positive cells) ± SEM of three independent experiments. ( C to G ) IMR90 ER:RAS cells were treated with 4OHT and repeatedly transfected with indicated siRNA and pooled nontarget siRNA control. siTP53 was used as a positive control. (C) After 5 days of treatment, a BrdU incorporation assay was conducted. Results are expressed as means ± SEM of three independent experiments. (D) Total DAPI-stained nuclei counted by high-content analysis at 8 days. Results are expressed as means ± SEM of three independent experiments. (E) After 10 days, SA-β-Gal activity assay was conducted. Scale bars, 100 μm. (F) qRT-PCR analysis of CDKN1A , CDKN2A , and CDKN2B transcripts. Results are expressed as means ± SEM of three independent experiments. (G) Western blot for p53 expression at 8 days. All statistical significance was calculated using one-way analysis of variance (ANOVA). *** P < 0.001, ** P < 0.01, and * P < 0.05.

Article Snippet: TLR2 complementary DNA (cDNA) was amplified from the pcDNA3-TLR2-YFP plasmid (Addgene, 13016) using primers flanked with Xho I sites.

Techniques: Infection, Expressing, Plasmid Preparation, Control, Staining, Transfection, Positive Control, BrdU Incorporation Assay, High Content Screening, Activity Assay, Quantitative RT-PCR, Western Blot

Journal: Science Advances

Article Title: The innate immune sensor Toll-like receptor 2 controls the senescence-associated secretory phenotype

doi: 10.1126/sciadv.aaw0254

Figure Lengend Snippet: ( A ) Heat map showing the relative fold change of acute-phase response transcripts of samples from the acute-phase response gene set from the mRNA transcriptomes. Transcriptome analysis (AmpliSeq) was performed in mRNA from IMR90 ER:RAS cells transfected with pooled siRNA for TLR2 and TLR10 and nontarget pool as a control. Genes with significant changes between nontarget siRNA control and both TLR2 and TLR10 knockdown are in bold characters. Adjusted P values were calculated using Benjamini and Hochberg false discovery rate of three independent experiments. Bold genes represent adjusted P < 0.05. ( B ) qRT-PCR validation of acute-phase response targets from samples obtained similarly to (A). Results are expressed as means ± SEM of three independent experiments. Statistical significance was calculated using one-way ANOVA and Dunnett’s multiple comparisons tests. *** P < 0.001, ** P < 0.01, and * P < 0.05. ( C ) qRT-PCR analysis of A-SAA expression in IMR90 ER:RAS and ER:STOP cells with up to 10 days of 4OHT treatment. ( D ) IMR90 ER:RAS cells were treated with 4OHT and repeatedly transfected with pooled siRNA targeting SAA1 and SAA2 and nontarget siRNA as control for 8 days. Western blot of the conditioned medium for indicated antibodies. ( E ) IMR90 cells transfected with pooled siRNA for TLR2 and TLR10 were treated with A-SAA (10 μg/ml) for 3 hours, and qRT-PCR was performed to measure IL1 β expression. Results are expressed as means ± SEM of three independent experiments. ( F ) Immunofluorescence staining and quantification of IL-1β expression by high-content analysis. Scale bars, 250 μm. ( G ) qRT-PCR for IL1 α, IL1 β, IL6 , and IL8 expression. Results are expressed as means ± SEM of three independent experiments. ( H ) IMR90 ER:RAS cells were transfected with siSAA2 and treated with 1 μm Pam2CSK4 for 5 days. qRT-PCR of IL1 α, IL1 β, and IL6 expression. ( I ) Heat map showing TLR2 SAA1 and SAA2 expression in available transcriptomic data from adriamycin (ADR) mediated therapy-induced senescence (TIS) lymphoma cells (GSE31099), OIS mediated by mutant BRAF in human melanocytes (OIS) (GSE46801), stasis in human mammary epithelial cells (HMEC) (GSE16058), DNA damage-induced senescence in BJ cells (DDR) (GSE13330), replicative senescence in BJ cells (replicative) (GSE13330), and developmental senescence in the mesonephros (developmental) (GSE49108). ( J ) GSEA plots for the 267 genes regulated coregulated by TLR2 and TLR10 in OIS (fig. S2H) in the transcriptomes from (I). All statistical significance was calculated using one-way ANOVA. *** P < 0.001, ** P < 0.01, and * P < 0.05.

Article Snippet: TLR2 complementary DNA (cDNA) was amplified from the pcDNA3-TLR2-YFP plasmid (Addgene, 13016) using primers flanked with Xho I sites.

Techniques: Transfection, Control, Knockdown, Quantitative RT-PCR, Biomarker Discovery, Expressing, Western Blot, Immunofluorescence, Staining, High Content Screening, Mutagenesis

Journal: Science Advances

Article Title: The innate immune sensor Toll-like receptor 2 controls the senescence-associated secretory phenotype

doi: 10.1126/sciadv.aaw0254

Figure Lengend Snippet: ( A and B ) IMR90 ER:RAS cells were treated with 4OHT and repeatedly transfected with indicated pooled siRNA and nontarget siRNA as control for 8 days. TLR2 , SAA1 , and SAA2 transcripts were measured by qRT-PCR. Results are expressed as means ± SEM of three independent experiments. ( C ) IMR90 cells were transfected with siRNA targeting RELA, IRF3, TLR2, and STING for 2 days, followed by transfection with 2.5 μg of herrings-testes DNA (HT-DNA) for 24 hours. TLR2 transcripts were measured by qRT-PCR. Results are expressed as means ± SEM of three independent experiments. ( D ) Western blot for STING dimerization. HT-DNA transfection of IMR90 cells were used as positive control for STING dimerization. IMR90 ER:RAS cells were transfected with siRNA targeting TLR2, TLR10, and STING for 8 days with 4OHT. All statistical significance was calculated using a one-way ANOVA. *** P < 0.001, ** P < 0.01, and * P < 0.05.

Article Snippet: TLR2 complementary DNA (cDNA) was amplified from the pcDNA3-TLR2-YFP plasmid (Addgene, 13016) using primers flanked with Xho I sites.

Techniques: Transfection, Control, Quantitative RT-PCR, Western Blot, Positive Control

Journal: Science Advances

Article Title: The innate immune sensor Toll-like receptor 2 controls the senescence-associated secretory phenotype

doi: 10.1126/sciadv.aaw0254

Figure Lengend Snippet: ( A ) Representative IHC staining and IHC score quantification of IL-1α in PanIN generated in tlr2 +/+ or tlr2 −/− Pdx-Cre Kras G12D mice. Scatter plot represents the value for individual animals (dots), and the horizontal line represents group means ( n = 5) ± SEM. Statistical significance was calculated using one-tailed Student’s t test. * P < 0.05 ( B ) qRT-PCR results for SASP factors IL-1β, IL-1α, and IL-6 from liver samples from WT and tlr2 −/− mice 6 days after receiving hydrodynamic delivery of Nras G12V/D38A negative control or oncogenic Nras G12V transposon as indicated. Scatter plot represents the value for individual animals (dots), and the horizontal line represents group means ( n = 3) ± SEM. Statistical significance was calculated using two-tailed students t test. * P < 0.05 and ** P < 0.01. ( C ) Representative IHC staining for Nras, Tlr2, IL-1β, p21, and Biotin-SBB in corresponding liver sections from mice in (B). Scale bars, 50 μm.

Article Snippet: TLR2 complementary DNA (cDNA) was amplified from the pcDNA3-TLR2-YFP plasmid (Addgene, 13016) using primers flanked with Xho I sites.

Techniques: Immunohistochemistry, Generated, One-tailed Test, Quantitative RT-PCR, Negative Control, Two Tailed Test

Journal: PloS one

Article Title: Stiffening-induced high pulsatility flow activates endothelial inflammation via a TLR2/NF-κB pathway.

doi: 10.1371/journal.pone.0102195

Figure Lengend Snippet: Figure 2. Proximal stiffening induces proinflammatory response and upregulates TLR2 expression in downstream bovine PAECs. (A-B) High pulsatility flow (HPF), due to the use of a “stiff” tube upstream to cell culture, upregulated proinflammatory molecule mRNA (ICAM-1, VCAM-1, MCP-1 and E-selectin) and protein (MCP-1) in healthy PAECs, compared to low pulsatility flow (LPF) or to static conditions. (C-D) The mRNA and protein expression of TLR2 but not TLR4 in PAECs was highly upregulated by HPF. *: p<0.05 versus Static, †: p<0.05 versus LPF. https://doi.org/10.1371/journal.pone.0102195.g002

Article Snippet: Tissue cryosections were similarly stained with human TLR2 (1∶50 dilution; Imgenex).

Techniques: Expressing, Cell Culture

Journal: PloS one

Article Title: Stiffening-induced high pulsatility flow activates endothelial inflammation via a TLR2/NF-κB pathway.

doi: 10.1371/journal.pone.0102195

Figure Lengend Snippet: Figure 3. Pharmacological or siRNA inhibition of TLR2 results in suppression of PAEC proinflammatory responses caused by stiffening-induced HPF. (A, C) At the mRNA level, TLR2/4 inhibitor OxPAPC or TLR2 siRNA but not TLR4 inhibitor CLI-095, decreased PAEC expression of ICAM-1, VCAM-1, MCP-1 and E-selectin mRNAs under HPF; TLR4 siRNA decreased ICAM-1 and E-selectin but not VCAM-1 and MCP-1 mRNAs. “*”: p<0.05 versus LPF, “†”: p<0.05 versus HPF. (B, D) At the protein level, the MCP-1 expression in PAECs exposed to HPF was inhibited by OxPAPC or TLR2 siRNA treatment but not CLI-095 or TLR4 siRNA. The black line in the blot images (D, right) shows separated lanes obtained on the same gel. https://doi.org/10.1371/journal.pone.0102195.g003

Article Snippet: Tissue cryosections were similarly stained with human TLR2 (1∶50 dilution; Imgenex).

Techniques: Inhibition, Expressing

Journal: PloS one

Article Title: Stiffening-induced high pulsatility flow activates endothelial inflammation via a TLR2/NF-κB pathway.

doi: 10.1371/journal.pone.0102195

Figure Lengend Snippet: Figure 4. Activation of TLR2/NF-κB pathway mediated pro-inflammatory responses of PAECs exposed to stiffening-induced HPF. Pulse flow modulates TLR2-induced NF-κB activation in BPAECs. (A) Representative fluorescent images and quantitative measures of NF-kBp65 staining (red) in PAECs show HPF stimulation of PAECs led to increased intranuclear translocation or activation of NF-κB, which was reduced by TLR2/4 inhibitor OxPAPC and TLR2 siRNA. Blue stains show the nuclei. The scale bar shows 50 µm. (B) HPF stimulation of PAECs increased the mRNA levels of IKKα and IKKβ, both of which were attenuated in siRNA-transfected cells with knockdown of TLR2. (C) NF-κB inhibitor (BAY 11–7082) decreased the MCP-1 expression by PAECs exposed to HPF. *: p<0.05 versus LPF, †: p<0.05 versus HPF. https://doi.org/10.1371/journal.pone.0102195.g004

Article Snippet: Tissue cryosections were similarly stained with human TLR2 (1∶50 dilution; Imgenex).

Techniques: Activation Assay, Staining, Translocation Assay, Transfection, Knockdown, Expressing

Journal: PloS one

Article Title: Stiffening-induced high pulsatility flow activates endothelial inflammation via a TLR2/NF-κB pathway.

doi: 10.1371/journal.pone.0102195

Figure Lengend Snippet: Figure 5. The circulating media from the cells exposed to HPF upregulate the inflammatory responses in normal PAECs through the TLR2/NF-κB pathway. The conditioned circulating media collected from cell cultures under the HPF and LPF conditions, respectively labeled as LPF- FM and HPF-FM, were used to culture normal PAECs for 24 h. (A) The mRNA expressions of ICAM-1, VCAM-1, MCP-1 and E-selectin were upregulated by HPF-FM condition, which were then reduced by OxPAPC and siTLR2. (B) Similar changes were shown in the MCP-1 protein expression. (C) NF-κB intranuclear translocation increased in PAECs stimulated with HPF- FM, which was reduced by OxPAPC and siTLR2. *: p<0.05 versus LPF-FM, †: p<0.05 versus HPF-FM. https://doi.org/10.1371/journal.pone.0102195.g005

Article Snippet: Tissue cryosections were similarly stained with human TLR2 (1∶50 dilution; Imgenex).

Techniques: Labeling, Expressing, Translocation Assay

Journal: PloS one

Article Title: Stiffening-induced high pulsatility flow activates endothelial inflammation via a TLR2/NF-κB pathway.

doi: 10.1371/journal.pone.0102195

Figure Lengend Snippet: Figure 6. Enhanced TLR and MCP-1 expression in the distal pulmonary artery endothelium in vivo. (A) PAECs from calves with hypoxia-induced pulmonary hypertension (PH) show elevated expression of TLR2 and TLR4, compared to control (CO). *:p<0.05. (B) Both immunostaining and western blotting results show elevated MCP-1 expression by PH-ECs compared to CO-ECs from calves. “PA” indicates the lumen of a pulmonary artery. *:p<0.05. (C) Enhanced TLR2 expression in the pulmonary arterial endothelium of human with pulmonary arterial hypertension (PAH). Cryosections of human intra-lobar pulmonary arteries were immunostained with TLR2 (red fluorescence) and counterstained with DAPI (cell nuclei, blue). Elastic lamellae showed green auto-fluorescence. https://doi.org/10.1371/journal.pone.0102195.g006

Article Snippet: Tissue cryosections were similarly stained with human TLR2 (1∶50 dilution; Imgenex).

Techniques: Expressing, In Vivo, Control, Immunostaining, Western Blot