tlr2 complementary dna cdna  (Addgene inc)

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