Journal: Frontiers in Immunology

Article Title: Toll-Like Receptor 8 Is a Major Sensor of Group B Streptococcus But Not Escherichia coli in Human Primary Monocytes and Macrophages

doi: 10.3389/fimmu.2017.01243

Figure Lengend Snippet: IFN regulatory factor 5 (IRF5) is activated by TLR8 ligands and cytosolic dsDNA sensing, IRF3 is activated by TLR4 signaling and cytosolic dsDNA and dsRNA sensors, while only TLR8 is antagonized by TLR2 ligand. (A) Monocytes were stimulated with CL75 (1 μg/ml), LPS (K12, 100 ng/ml), pdA:dT (dsDNA, 1 μg/ml), or poly(I:C) (dsRNA, 1 μg/ml). The nucleic acid ligands were transfected using L2K. Fixation was done at 30, 60, and 120 min poststimulation and IF staining of IRF5 and IRF3 was performed. (B) Monocytes were stimulated with the TLR8 agonist polyuridylic acid (pU) in complex with poly- l -arginine (pLA) or with pdA:dT/L2K. The ligands (1 μg/ml) were added alone or together with FLS-1 (100 ng/ml), and cells were fixed and stained after 120 min of incubation. Quantification of nuclear accumulation was done by high-content screening (Scan^R, 20×). Significance was tested relative to no stimuli (NS). n = 4.

Article Snippet: The following antibodies were used: mouse antihuman IRF5 mAb (Abcam, #10T1), rabbit antihuman IRF3 XP mAb [Cell Signaling Technology (CST), no. 11904], rabbit antihuman p65/RelA XP mAb (CST, no. 8242), rabbit antihuman p65 A (Santa Cruz Biotechnology, no. sc-109), rabbit antihuman IRF1 XP mAb (CST, no. 8478), and rabbit antihuman TLR8 XP mAb (CST, no. D3Z6J).

Techniques: Transfection, Staining, Incubation, High Content Screening

Journal: Frontiers in Immunology

Article Title: Toll-Like Receptor 8 Is a Major Sensor of Group B Streptococcus But Not Escherichia coli in Human Primary Monocytes and Macrophages

doi: 10.3389/fimmu.2017.01243

Figure Lengend Snippet: Activation of IFN regulatory factor 5 (IRF5) by TLR8 ligand and Group B Streptococcus (GBS) is antagonized by TLR2 costimulation, while activation of IRF5 by viable Escherichia coli is not. Monocytes were stimulated with CL75 (1 μg/ml), viable or heat-inactivated (HI) GBS (MOI 1.0) and E. coli (MOI 2.0), and with or without FSL-1 costimulation (100 ng/ml) for 2 h. The monocytes were subsequently fixed and double IF stained IRF5 and p65/RelA. Nuclear accumulation was quantified by high-content screening (Scan^R, 20×). NS, no stimuli. nd, not done. n = 4.

Article Snippet: The following antibodies were used: mouse antihuman IRF5 mAb (Abcam, #10T1), rabbit antihuman IRF3 XP mAb [Cell Signaling Technology (CST), no. 11904], rabbit antihuman p65/RelA XP mAb (CST, no. 8242), rabbit antihuman p65 A (Santa Cruz Biotechnology, no. sc-109), rabbit antihuman IRF1 XP mAb (CST, no. 8478), and rabbit antihuman TLR8 XP mAb (CST, no. D3Z6J).

Techniques: Activation Assay, Staining, High Content Screening

Journal: Frontiers in Immunology

Article Title: Toll-Like Receptor 8 Is a Major Sensor of Group B Streptococcus But Not Escherichia coli in Human Primary Monocytes and Macrophages

doi: 10.3389/fimmu.2017.01243

Figure Lengend Snippet: TLR8 accumulates on Group B Streptococcus (GBS) phagosomes in macrophages and monocytes. (A) THP-1 TLR8-KO cells were transduced with TLR8 mNeonGreen construct (TLR8mNG) and differentiated with PMA. Macrophages were infected with viable GBS (MOI 1.0) prestained with Alexa 647. Gentamycin was added 1 h postinfection and cells were fixed with paraformaldehyde after 20 h. DNA was stained with Hoechst 33342. (B) Primary monocytes were infected with viable GBS (MOI 10), and Gentamycin was added 1 h postinfection. Cells were fixed with paraformaldehyde after 3 h and TLR8 was IF stained with a mAb. DNA was stained with Hoechst 33342 in PBS/saponin to visualize GBS phagosomes (arrowhead) and monocyte nuclei. Images were acquired using Olympus Scan^R (60×). Scale bars are 10 µm.

Article Snippet: The following antibodies were used: mouse antihuman IRF5 mAb (Abcam, #10T1), rabbit antihuman IRF3 XP mAb [Cell Signaling Technology (CST), no. 11904], rabbit antihuman p65/RelA XP mAb (CST, no. 8242), rabbit antihuman p65 A (Santa Cruz Biotechnology, no. sc-109), rabbit antihuman IRF1 XP mAb (CST, no. 8478), and rabbit antihuman TLR8 XP mAb (CST, no. D3Z6J).

Techniques: Transduction, Construct, Infection, Staining

Journal: Frontiers in Immunology

Article Title: Toll-Like Receptor 8 Is a Major Sensor of Group B Streptococcus But Not Escherichia coli in Human Primary Monocytes and Macrophages

doi: 10.3389/fimmu.2017.01243

Figure Lengend Snippet: Group B Streptococcus (GBS) trigger cytokine production in part via TLR8-IRF5 signaling, while Escherichia coli induces cytokines independently of TLR8, yet partly dependent on IRF5. Monocyte-derived macrophages (MDM) were transfected with siRNA against TLR7, TLR8, IRF5, and STING, as indicated. Following successful silencing the cells were stimulated with pU/pLA or infected with viable GBS wt (MOI 1.0) or E. coli (MOI 2.0). Cytokine induction relative to non-stimulated cells after 4 h of infection was determined by quantitative real-time PCR (qPCR). Significance of gene silencing was tested in relation to non-targeting control (NTC). n = 5.

Article Snippet: The following antibodies were used: mouse antihuman IRF5 mAb (Abcam, #10T1), rabbit antihuman IRF3 XP mAb [Cell Signaling Technology (CST), no. 11904], rabbit antihuman p65/RelA XP mAb (CST, no. 8242), rabbit antihuman p65 A (Santa Cruz Biotechnology, no. sc-109), rabbit antihuman IRF1 XP mAb (CST, no. 8478), and rabbit antihuman TLR8 XP mAb (CST, no. D3Z6J).

Techniques: Derivative Assay, Transfection, Infection, Real-time Polymerase Chain Reaction, Control

Journal: bioRxiv

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

doi: 10.1101/466755

Figure Lengend Snippet: ( A ) Schematic showing IMR90 ER:RAS cells treated 4OHT undergo OIS. IMR90 ER:Stop cells serve as a control and retain proliferative capacity with 4OHT. (B) 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 (upper panel). BrdU incorporation in IMR90 ER:RAS cells treated with 4OHT for up to 8 days as indicated. (D) Immunohistochemical staining for Nras in liver sections from WT mice 6 days following hydrodynamic delivery of Nras G12V/D38A (n=5) and Nras G12V (n=4) transposons. Scale bar 50µm. RNA was extracted from snap frozen liver samples and tlr2 mRNA expression was measured using qRT-PCR. Scatter plots represents value per animal with the horizontal line representing group mean ± SEM. Statistical significance was calculated using Students two-tailed t -test. **p<0.01.

Article Snippet: TLR2 was blocked by incubating cells with 10 µg/ml of anti-TLR2 (R&D, MAB2616) or IgG2B Isotype control.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, BrdU Incorporation Assay, Immunohistochemical staining, Staining, Two Tailed Test

Journal: bioRxiv

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

doi: 10.1101/466755

Figure Lengend Snippet: (A) Transcriptome analysis and Gene Set Enrichment Analysis was conducted to compare oncogene-induced senescent cells (IMR90 ER:RAS + 4OHT at 8 days) vs. control cells (IMR90 ER:Stop + 4OHT at 8 days) of 6 independent experiments. Adjusted p-value of significant gene sets related to the innate immune response and pattern recognition receptors are shown. (B) Conditioned medium (CM) generated by IMR90 ER:Stop and ER:RAS following 8 days of 4OHT treatment was transferred to proliferating IMR90 cells and cultured for 2 days. After a 16-hour pulse, BrdU incorporation was high content analysis. qRT-PCR of TLR2 mRNA in IMR90 cells treated with CM from ER:RAS and ER:Stop cells was assessed. Results expressed as mean ± SEM of 3 independent experiments. Statistical significance was calculated using Students two-tailed t -test. **p < 0.01, * p < 0.05. (C) TLR2 mRNA expression determined by qRT-PCR of IMR90 cells infected with RAS G12V vector and empty vector (EV) as control. (D) IMR90 cells were treated with 100 µM Etoposide. After 2 days, BrdU incorporation was assessed by immunofluorescence and qRT-PCR analysis for TLR2 expression was conducted. (D) Immunohistochemical staining of PanIN structures from Pdx-Cre Kras G12D for the expression of Tlr2 and Ki67 expression in consecutive sections. White arrows indicate PanIN structure. Black arrows indicate pancreatic acinar cells. (F) qRT-PCR analysis of the senescence markers dcr2 and arf and the key SASP factor il-1β from snap frozen liver samples from WT mice 6 days after receiving hydrodynamic delivery of Nras G12V/D38A (n=5) and Nras G12V (n=4) transposons respectively. Scatter plot represents value from individual animals and the horizontal line represents group mean ± SEM. Statistics: Students two-tailed t -test. ***p<0.001, ****p<0.0001. (G) Analysis of Tlr2 and p21 Cip-1 expression was conducted by immunohistochemistry in lung sections from wild type (wt) or nfkb1 knock out mice ( nfkb1 -/- ) at 9.5 months of age. 10-15 random images were captured per mouse and average percentage positivity calculated for airway epithelial compartments. Scatter plots represent mean percentage positivity for each animal with the horizontal line representing group median. Statistical significance was calculated using Mann Whitney U test. *p < 0.05, **p < 0.01. Representative images of p21 and TLR2 staining by immunohistochemistry (positive, brown; negative, blue) in airway epithelial cells from wt and nfkb1-/- mice. (E) Analysis of Tlr2 expression by immunohistochemistry in lung sections of wt mice at 6.5 months of age (Young) and 24 months of age week (old). Scatter plots were generated from 10-15 random images captured per animal with individual points representing mean percentage positivity for each mouse with horizontal line representing group median. Statistics: Mann-Whitney U test. *p < 0.05. Representative images of TLR2 staining by immunohistochemistry (positive, brown; negative, blue) in alveolar cells from wt mice 6.5 and 24 months of age.

Article Snippet: TLR2 was blocked by incubating cells with 10 µg/ml of anti-TLR2 (R&D, MAB2616) or IgG2B Isotype control.

Techniques: Generated, Cell Culture, BrdU Incorporation Assay, High Content Screening, Quantitative RT-PCR, Two Tailed Test, Expressing, Infection, Plasmid Preparation, Immunofluorescence, Immunohistochemical staining, Staining, Immunohistochemistry, Knock-Out, MANN-WHITNEY

Journal: bioRxiv

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

doi: 10.1101/466755

Figure Lengend Snippet: (A-F) IMR90 ER:RAS cells were treated with 4OHT and repeatedly transfected with indicated pooled siRNA targeting TLR1 subfamily receptors and non-target (NT) siRNA as control for 8 days. (A) qRT-PCR of TLR1, TLR2, TLR6, TLR10 expression to validate knockdown. (B) ELISA and western blot for IL-1β content in the conditioned medium following indicated TLR knockdown. (C) qPCR of TLR2 and TLR10 expression following the pooled and individual siRNA media knockdown of respective target genes. (D) Western blot of IL-1β protein levels following TLR2 and TLR10 knockdown using pooled (T2-P, T10-P) and individual (T10-2, T2-1, T2-2, T2-3, T2-4) siRNA. (E) qRT-PCR of IL1B mRNA expression following TLR10 knockdown using pooled (T10-P) and individual (T10-2, T10-4) siRNA. (F) Representative images of immunofluorescence and high content analysis of IL6, IL8 and IL-1β expression in TLR2 and TLR10 knockdown cells. (G) Conditioned medium (CM) generated by IMR90 ER:Stop and ER:RAS following 8 days of 4OHT treatment was transferred to proliferating IMR90 cells with siRNA knockdown of TLR2 and TLR10 and cultured for 2 days. Immunofluorescence and high content analysis was used to determine IL-1α, IL-1β, IL-6 and IL-8 protein levels in the IMR90 cells. Results expressed as mean ± SEM of 3 independent experiments. Statistical significance was calculated using One-Way ANOVA and Dunnett’s multiple comparison’s tests. ***p < 0.001, **p < 0.01, * p < 0.05, NS, non-significant. (H) Transcriptome analysis (Ampliseq) of IMR90 ER:RAS cells transfected with pooled siRNA for TLR2 and TLR10, and non-target (NT) as control. Table identifies the total number of genes significantly regulated by TLR2 and TLR10 during OIS. Heat-map showing sample clustering.

Article Snippet: TLR2 was blocked by incubating cells with 10 µg/ml of anti-TLR2 (R&D, MAB2616) or IgG2B Isotype control.

Techniques: Transfection, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Immunofluorescence, High Content Screening, Generated, Cell Culture

Journal: bioRxiv

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

doi: 10.1101/466755

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. Non-target pooled siRNA (NT) was used as control. Representative images are shown. Scale bar 250 µm. (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. Non-target pooled siRNA (NT) was used as control. Western blot against b-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 expressed as mean ± SEM of 3 independent experiments. (D) Venn diagram shows the number of genes that are significantly induced by TLR2 and TLR10 during OIS, and the intersection represents the number of genes regulated by both TLR2 and TLR10. This signature of 267 genes will be used for gene set enrichment analysis in additional senescence transcriptomes in and and S7). (E) Top regulated terms identified through of coregulated genes in (H) using DAVID gen ontology analysis. Chart bars represents 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 IKK α/β and p38 MAPK was performed. (I) IMR90 ER:RAS cells were treated with 4OHT and repeatedly transfected with indicated pooled siRNA and non-target (NT) siRNA as control for 5 days. Western blots were conducted for phosphorylation of p65 and total p65 protein levels.

Article Snippet: TLR2 was blocked by incubating cells with 10 µg/ml of anti-TLR2 (R&D, MAB2616) or IgG2B Isotype control.

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

Journal: bioRxiv

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

doi: 10.1101/466755

Figure Lengend Snippet: ( A ) IMR90 ER:RAS and ER:Stop cells were treated with 10 μM of IKK inhibitor (BAY 11-07082) and p38 MAPK inhibitor (SB202190). A BrdU incorporation assay was conducted and analysed by high content microscopy. (B) CDKN1A mRNA expression was measured by qRT-PCR in sample treated as in (A). (C) Crystal violet staining of the cells 12 days after siRNA transfection and 4OHT treatment of IMR90 ER:RAS cells. (D) Total cell count (nuclei count number) in the total surface of tissue culture wells by high content analysis of IMR90 ER:RAS cells transfected with indicated siRNA for 12 days. (E) BrdU incorporation assay following pooled siRNA knockdown of all TLR family members at 5 days of 4OHT treatment in IMR90 ER:RAS and IMR90 ER:Stop cells. Results expressed as mean ± SEM of 3 independent experiments. Statistical significance was calculated using two-tailed Students t-tests. ***p < 0.001, **p < 0.01, * p < 0.05, n.s., non-significant. (F) Conditional medium (CM) generated by IMR90 ER:Stop and ER:RAS following 8 days of 4OHT treatment was transferred to proliferating IMR90 cells with siRNA knockdown of TLR2 and TLR10 and cultured for 2 days. Immunofluorescence and high content analysis was used to determine BrdU incorporation in the IMR90 cells. Results expressed as mean ± SEM of 3 independent experiments. Statistical significance was calculated using One-Way ANOVA. ***p < 0.001, **p < 0.01, * p < 0.05, ns, non-significant.

Article Snippet: TLR2 was blocked by incubating cells with 10 µg/ml of anti-TLR2 (R&D, MAB2616) or IgG2B Isotype control.

Techniques: BrdU Incorporation Assay, Microscopy, Expressing, Quantitative RT-PCR, Staining, Transfection, Cell Counting, High Content Screening, Two Tailed Test, Generated, Cell Culture, Immunofluorescence

Journal: bioRxiv

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

doi: 10.1101/466755

Figure Lengend Snippet: (A) IMR90 cells infected with TLR2 or H-Ras G12V expression vectors, or empty vector control (EV) were seeded at low density and stained with crystal violet after 2 weeks. The staining was quantified to obtain relative cell content. Results expressed as mean ± SEM of 3 independent experiments. (B) SA-β-GAL staining was carried out on TLR2 and HRas G12V expressing cells. Results expressed as mean (% positive cells) ± SEM of 3 independent experiments. (C-G) IMR90 ER:RAS cells were treated with 4OHT and repeatedly transfected with indicated siRNA and pooled non-target (NT) siRNA control. siTP53 was used as a positive control. (C) After 5 days of treatment, a BrdU incorporation assay was conducted. Results expressed as mean ± SEM of 3 independent experiments. (D) Total DAPI stained nuclei counted by high content analysis at 8 days. Results expressed as mean ± SEM of 3 independent experiments. (E) After 10 days SA-β-GAL activity assay was conducted. Scale bar 100 mm. (F) qRT-PCR analysis of CDKN1A, CDKN2A and CDKN2B transcripts. Results expressed as mean ± SEM of 3 independent experiments. (G) Western blot for p53 expression at eight days. All statistical significance was calculated using One-Way ANOVA. ***p < 0.001, **p < 0.01, * p < 0.05, ns, non-significant.

Article Snippet: TLR2 was blocked by incubating cells with 10 µg/ml of anti-TLR2 (R&D, MAB2616) or IgG2B Isotype control.

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

Journal: bioRxiv

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

doi: 10.1101/466755

Figure Lengend Snippet: (A) Representative immunohistochemical (IHC) staining and IHC score quantification of IL-1α in PanIN generated in tlr2 +/+ or tlr2 -/- Pdx-Cre Kras G12D mice. Scatter plot represents value for individual animals (dots) and the horizontal line represents group mean (n=5) ± SEM. Statistical significance was calculated using one-tailed students t -test. *p<0.05 (B) Representative IHC staining for Nras (top row) and IL-1β (bottom row) in liver sections from wild-type (WT) and tlr2 -/- mice 6 days after receiving hydrodynamic delivery of Nras G12V/D38A negative control or oncogenic Nras G12V transposon as indicated. Image magnification is shown in the il-1β box insert. Scale bar 50µm (C) qRT-PCR results for SASP factors IL-1β, IL-1α and IL-6 from liver samples from corresponding mice in (B). Scatter plot represents value for individual animals (dots) and the horizontal line represents group mean (n=3) ± SEM. Statistical significance was calculated using two-tailed students t -test. *p<0.05, **p<0.01. ns, non-significant.

Article Snippet: TLR2 was blocked by incubating cells with 10 µg/ml of anti-TLR2 (R&D, MAB2616) or IgG2B Isotype control.

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

Journal: bioRxiv

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

doi: 10.1101/466755

Figure Lengend Snippet: (A) Gene Set Enrichment Analysis (GSEA) of the transciptome from Supplemental Fig. S2H showing top two regulated gene set groups, and GSEA enrichment plot of genes of the acute phase response in TLR2 siRNA transfected IMR90 ER:RAS 4OHT induced cells. (B) IMR90 ER:RAS and ER:Stop were treated with 4OHT for up to 10 days with RNA collected at indicated intervals. qRT-PCR for SAA1 mRNA expression (C) qRT-PCR for SAA1 and SAA2 mRNA expression in TLR2 overexpressing IMR90 cells activated with 1 µg/ml Pam2CSK4 ligand for 3 hours. Results expressed as mean ± SEM of 3 independent experiments. (D) Confirmation of SAA1 and SAA2 expression knockdown by qRT-PCR in IMR90 ER:RAS and ER:Stop cells transfected with pooled siRNA targeting SAA1 and SAA2, and non-target (NT) control and treated with 4OHT for 8 days. Results expressed as mean ± SEM of 3 independent experiments. All statistical significance was calculated using One-Way ANOVA. ***p < 0.001, **p < 0.01, * p < 0.05, ns, non-significant. (E) qRT-PCR for Cdkn1a (p21), Cdkn2a (p16) and Saa2 expression in whole lung tissue from wt or nfkb1-/- mice at 9.5 months of age. Dot plots represent the ΔΔCT value for individual animals generated by normalizing to 18S expression. The horizontal line represents group median. Statistical significance was calculated using Mann-Whitney U test. **p < 0.01. (F) Immunohistochemical staining of PanIN structures from Pdx-Cre Kras G12D for the expression of Tlr2, Saa1 and Ki67 expression in consecutive sections. White arrows indicate PanIN structure. Black arrows indicate pancreatic acinar cells.

Article Snippet: TLR2 was blocked by incubating cells with 10 µg/ml of anti-TLR2 (R&D, MAB2616) or IgG2B Isotype control.

Techniques: Transfection, Quantitative RT-PCR, Expressing, Generated, MANN-WHITNEY, Immunohistochemical staining, Staining

Journal: bioRxiv

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

doi: 10.1101/466755

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 non-target pool (NT) as a control. Genes with significant changes between NT siRNA control and both TLR2 and TLR10 knockdown are in bold characters. Adjusted p-values were calculated using Benjamini and Hochberg (BH) false discovery rate of 3 independent experiments. Bold genes represent adjusted p-value < 0.05. (B) qRT-PCR validation of acute phase response targets from samples obtained similarly to (A). Results expressed as mean ± SEM of 3 independent experiments. Statistical significance was calculated using One-Way ANOVA and Dunnett’s multiple comparison’s tests. ***p < 0.001, **p < 0.01, * p < 0.05, NS, non-significant. (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 non-target (NT) 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 10 µg/ml A-SAA for 3 hours and qRT-PCR was performed to measure IL1B expression. Results are expressed as mean ± SEM of 3 independent experiments. (F) Immunofluorescence staining and quantification of IL-1β expression by high content analysis. Scale bar 250 µm. (G) qRT-PCR for IL1A , IL1B, IL6, IL8 expression. Results expressed as mean ± SEM of 3 independent experiments. (H) Heatmap showing TLR2 SAA1 and SAA2 expression in available transcriptomic data from Therapy-induced senescence lymphoma cells (TIS) (GSE31099), Oncogene-induced senescence mediated by mutant BRAF in human melanocytes (OIS) (GSE46801), Stasis in HMEC (GSE16058), DNA damage induced senescence in BJ cells (DDR) (GSE13330), Replicative senescence in BJ cells (replicative) (GSE13330) and developmental senescence in the mesenephos (developmental) (GSE49108). (I) Gene-set enrichment analysis plots for the 267 genes regulated co-regulated by TLR2 and TLR10 in OIS in the transcriptomes from (I). All statistical significance was calculated using One-Way ANOVA. ***p < 0.001, **p < 0.01, * p < 0.05, ns, non-significant.

Article Snippet: TLR2 was blocked by incubating cells with 10 µg/ml of anti-TLR2 (R&D, MAB2616) or IgG2B Isotype control.

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

Journal: bioRxiv

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

doi: 10.1101/466755

Figure Lengend Snippet: (A) IL1A and IL1B mRNA expression was assessed by qRT-PCR in TLR2 expressing and non-expressing control (EV) IMR90 cells treated with 1 µM A-SAA or 1 µM Pam2CSK4 for 3 hours. Results expressed as mean ± SEM of 3 independent experiments. (B) IL1A, IL1B, IL6 and IL8 mRNA expression was assessed by qRT-PCR in TLR2 expressing and non-expressing control (EV) IMR90 cells treated with 10 µg/ml A-SAA for 3 hours. Results expressed as mean ± SEM of 3 independent experiments. (C) IMR90 cells expressing TLR2 or infected with empty vector (EV) as a control were incubated for 3 hours with recombinant active SAA (A-SAA) protein (10 µg/ml) or BSA (10 µg/ml) as a control. Western blot to measure IL-1β IL6 and IL8 protein levels as indicated. (D) TLR2 overexpressing IMR90 cells were treated with 10 µg/ml A-SAA plus 10 µg/ml of neutralising antibody against TLR2 for 2 hours. Assessment of IL1A, IL1B, IL6 and IL8 mRNA expression was conducted by qRT-PCR. Results expressed as mean ± SEM of 3 independent experiments. (E) IMR90 cells transfected with pooled siRNA for TLR2 and TLR10 were treated with 1 µg/ml A-SAA for 3 hours and IL6 and IL1A mRNA expression determined by qRT-PCR. Results expressed as mean ± SEM of 3 independent experiments. All statistical significance was calculated using One-Way ANOVA. ***p < 0.001, **p < 0.01, * p < 0.05, ns, non-significant. (F) qRT-PCR to confirm the knockdown efficiency of the indicated target gene by pooled siRNA. (G) IMR90 ER:RAS cells were treated with 4OHT and repeatedly transfected with indicated pooled siRNA and non-target (NT) siRNA as control for 8 days. ELISA for IL-1β levels in conditioned medium. Results expressed as mean ± SEM of 3 independent experiments. (F) GSEA plots of the gene set from in the transcriptome of replicative senescence BJ cells and programmed senescent cells of the mesonephros in .

Article Snippet: TLR2 was blocked by incubating cells with 10 µg/ml of anti-TLR2 (R&D, MAB2616) or IgG2B Isotype control.

Techniques: Expressing, Quantitative RT-PCR, Infection, Plasmid Preparation, Incubation, Recombinant, Western Blot, Transfection, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

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

doi: 10.1101/466755

Figure Lengend Snippet: ( A, B ) IMR90 ER:RAS cells were treated with 4OHT and repeatedly transfected with indicated pooled siRNA and non-target (NT) siRNA as control for 8 days. TLR2, SAA1 and SAA2 transcripts were measured by qRT-PCR. Results expressed as mean ± SEM of 3 independent experiments. (C) IMR90 cells were transfected with siRNA targeting TLR2 and STING for 2 days followed by transfection with 2.5 µg herrings-testes DNA (HT-DNA) for 24 hours. TLR2 transcripts were measured by qRT-PCR. Results are expressed as mean ± SEM of 3 independent experiments. (D) Western blot for STING dimerization. HTDNA 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, * p < 0.05, ns, non-significant.

Article Snippet: TLR2 was blocked by incubating cells with 10 µg/ml of anti-TLR2 (R&D, MAB2616) or IgG2B Isotype control.

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

Journal: bioRxiv

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

doi: 10.1101/466755

Figure Lengend Snippet: (A) GSEA enrichment plot of genes regulated by TLR2 and TLR10 in OIS described in in the transcriptome of PanIN cells compared to acinar normal cells (GSE33323). (B) Model of activation of the SASP by A-SAA-TLR2 in OIS.

Article Snippet: TLR2 was blocked by incubating cells with 10 µg/ml of anti-TLR2 (R&D, MAB2616) or IgG2B Isotype control.

Techniques: Activation Assay