Structured Review

InvivoGen r837
M45 inhibits IκBα degradation, TNFα and IL-6 production during MCMV infection. (A) NIH-3T3 cells were infected with wt MCMV-GFP, an M45 deletion mutant (ΔM45) or a revertant virus (RM45) at an MOI of 10 and treated 5 h postinfection with the TLR2 agonist Pam 3 CSK 4 (Pam., 0.1 µg/ml), the TLR4 agonist LPS (10 µg/ml), or IL-1β (20 ng/ml) for the indicated times. Levels of the indicated proteins were analyzed by immunoblotting. (B) BMDMs were infected with wt MCMV-GFP, ΔM45, or RM45 at an MOI of 3 and stimulated 8 h postinfection for 16 h with the TLR7 agonist R848 (0.1 µM). TNFα and IL-6 levels in the supernatant were determined by ELISA (mean ± SD). (C) RAW264.7 macrophages were infected with wt MCMV-GFP or ΔM45 at an MOI of 0.1, stimulated 24 h postinfection for 4 hours with TLR agonists Pam 3 CSK 4 (Pam.) or Malp-2 (TLR2), LPS (TLR4), <t>R837</t> (TLR7), or CpG (TLR9), in the presence of brefeldin A. Cells were fixed, permeabilized, and stained with a TNFα-specific antibody. The percentages of TNFα-positive cells within infected (GFP-positive) cell populations were determined by FACS analysis (mean ± SD).
R837, supplied by InvivoGen, used in various techniques. Bioz Stars score: 99/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Viral Mediated Redirection of NEMO/IKK? to Autophagosomes Curtails the Inflammatory Cascade"

Article Title: Viral Mediated Redirection of NEMO/IKK? to Autophagosomes Curtails the Inflammatory Cascade

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1002517

M45 inhibits IκBα degradation, TNFα and IL-6 production during MCMV infection. (A) NIH-3T3 cells were infected with wt MCMV-GFP, an M45 deletion mutant (ΔM45) or a revertant virus (RM45) at an MOI of 10 and treated 5 h postinfection with the TLR2 agonist Pam 3 CSK 4 (Pam., 0.1 µg/ml), the TLR4 agonist LPS (10 µg/ml), or IL-1β (20 ng/ml) for the indicated times. Levels of the indicated proteins were analyzed by immunoblotting. (B) BMDMs were infected with wt MCMV-GFP, ΔM45, or RM45 at an MOI of 3 and stimulated 8 h postinfection for 16 h with the TLR7 agonist R848 (0.1 µM). TNFα and IL-6 levels in the supernatant were determined by ELISA (mean ± SD). (C) RAW264.7 macrophages were infected with wt MCMV-GFP or ΔM45 at an MOI of 0.1, stimulated 24 h postinfection for 4 hours with TLR agonists Pam 3 CSK 4 (Pam.) or Malp-2 (TLR2), LPS (TLR4), R837 (TLR7), or CpG (TLR9), in the presence of brefeldin A. Cells were fixed, permeabilized, and stained with a TNFα-specific antibody. The percentages of TNFα-positive cells within infected (GFP-positive) cell populations were determined by FACS analysis (mean ± SD).
Figure Legend Snippet: M45 inhibits IκBα degradation, TNFα and IL-6 production during MCMV infection. (A) NIH-3T3 cells were infected with wt MCMV-GFP, an M45 deletion mutant (ΔM45) or a revertant virus (RM45) at an MOI of 10 and treated 5 h postinfection with the TLR2 agonist Pam 3 CSK 4 (Pam., 0.1 µg/ml), the TLR4 agonist LPS (10 µg/ml), or IL-1β (20 ng/ml) for the indicated times. Levels of the indicated proteins were analyzed by immunoblotting. (B) BMDMs were infected with wt MCMV-GFP, ΔM45, or RM45 at an MOI of 3 and stimulated 8 h postinfection for 16 h with the TLR7 agonist R848 (0.1 µM). TNFα and IL-6 levels in the supernatant were determined by ELISA (mean ± SD). (C) RAW264.7 macrophages were infected with wt MCMV-GFP or ΔM45 at an MOI of 0.1, stimulated 24 h postinfection for 4 hours with TLR agonists Pam 3 CSK 4 (Pam.) or Malp-2 (TLR2), LPS (TLR4), R837 (TLR7), or CpG (TLR9), in the presence of brefeldin A. Cells were fixed, permeabilized, and stained with a TNFα-specific antibody. The percentages of TNFα-positive cells within infected (GFP-positive) cell populations were determined by FACS analysis (mean ± SD).

Techniques Used: Infection, Mutagenesis, Enzyme-linked Immunosorbent Assay, Staining, FACS

2) Product Images from "PolyI:C-induced reduction in uptake of soluble antigen is independent of dendritic cell activation"

Article Title: PolyI:C-induced reduction in uptake of soluble antigen is independent of dendritic cell activation

Journal: International Immunology

doi: 10.1093/intimm/dxp053

Reduced uptake of soluble antigen by BMDC in the presence of TLR agonists. Granulocyte macrophage colony-stimulating factor (GM-CSF) BMDC were cultured for 4 h with OVA–Alexa555 (5 μg ml −1 ) in the presence of polyI:C (50 μg ml −1 ), CpG (0.5 μg ml −1 ), R837 (1 μg ml −1 ), LPS (1 μg ml −1 ) or left untreated. Cells were stained with anti-CD11c antibody and samples were acquired by flow cytometry. (A) The mean fluorescent intensity of cells for Alexa555 was determined after gating on the CD11c + cell population. The data of two independent representative experiments were pooled ( n = 4) and the standard deviation is indicated. (B) The frequency of OVA–Alexa555 + cells was determined after gating on the CD11c + cell population. The relative percentage of OVA–Alexa555 + BMDC in samples stimulated with TLR agonists is depicted in relation to the frequency of OVA–Alexa555 + BMDC in the absence of TLR stimulation (unst), which was set to 100%. The results of eight independent experiments were compiled and the standard deviation is indicated. (C) Mice were vaccinated intravenously with GM-CSF BMDC that had been pulsed overnight with OVA in the presence or absence of various TLR ligands. Seven days after vaccination, CFSE-labelled target cells were injected and antigen-specific lysis of peptide-pulsed targets cells versus unpulsed target cells was determined by flow cytometry the following day. The graph compiles data from two independent experiments ( n = 10). For statistical analysis, one-way analysis of variance in combination with Dunnett's (A and B) or Tukey's (C) multiple comparison test was used (** P
Figure Legend Snippet: Reduced uptake of soluble antigen by BMDC in the presence of TLR agonists. Granulocyte macrophage colony-stimulating factor (GM-CSF) BMDC were cultured for 4 h with OVA–Alexa555 (5 μg ml −1 ) in the presence of polyI:C (50 μg ml −1 ), CpG (0.5 μg ml −1 ), R837 (1 μg ml −1 ), LPS (1 μg ml −1 ) or left untreated. Cells were stained with anti-CD11c antibody and samples were acquired by flow cytometry. (A) The mean fluorescent intensity of cells for Alexa555 was determined after gating on the CD11c + cell population. The data of two independent representative experiments were pooled ( n = 4) and the standard deviation is indicated. (B) The frequency of OVA–Alexa555 + cells was determined after gating on the CD11c + cell population. The relative percentage of OVA–Alexa555 + BMDC in samples stimulated with TLR agonists is depicted in relation to the frequency of OVA–Alexa555 + BMDC in the absence of TLR stimulation (unst), which was set to 100%. The results of eight independent experiments were compiled and the standard deviation is indicated. (C) Mice were vaccinated intravenously with GM-CSF BMDC that had been pulsed overnight with OVA in the presence or absence of various TLR ligands. Seven days after vaccination, CFSE-labelled target cells were injected and antigen-specific lysis of peptide-pulsed targets cells versus unpulsed target cells was determined by flow cytometry the following day. The graph compiles data from two independent experiments ( n = 10). For statistical analysis, one-way analysis of variance in combination with Dunnett's (A and B) or Tukey's (C) multiple comparison test was used (** P

Techniques Used: Cell Culture, Staining, Flow Cytometry, Cytometry, Standard Deviation, Mouse Assay, Injection, Lysis

3) Product Images from "Anti-hepatitis B virus (HBV) response of imiquimod based toll like receptor 7 ligand in hbv-positive human hepatocelluar carcinoma cell line"

Article Title: Anti-hepatitis B virus (HBV) response of imiquimod based toll like receptor 7 ligand in hbv-positive human hepatocelluar carcinoma cell line

Journal: BMC Infectious Diseases

doi: 10.1186/s12879-017-2189-z

Status of epigenetic markers and transcription factors on TLR7 activation. HepG2.2.15 cells were treated with R837 for 72 h and then the cells were subjected to Western Blot for protein expression studies. a Protein expression of different histone modifications (H3K4Me3, H3K9Me3, H3K36Me3 and H3K9Ac) and p53 before and after TLR7 activation. b The relative expression of the proteins (H3K9Me3 and p53) in HepG2 and HepG2.2.15 cells
Figure Legend Snippet: Status of epigenetic markers and transcription factors on TLR7 activation. HepG2.2.15 cells were treated with R837 for 72 h and then the cells were subjected to Western Blot for protein expression studies. a Protein expression of different histone modifications (H3K4Me3, H3K9Me3, H3K36Me3 and H3K9Ac) and p53 before and after TLR7 activation. b The relative expression of the proteins (H3K9Me3 and p53) in HepG2 and HepG2.2.15 cells

Techniques Used: Activation Assay, Western Blot, Expressing

Flow cytometric analysis and alteration of key cell cycle regulators upon TLR7 activation. HepG2.2.15 cells were treated with R837 (8μg/ml) for 72 h and then subjected to flow cytometric analysis to study the status of G1/S arrest. a Cell cycle analysis by FACS. b Percentage of cells in different phases of cell cycle before and after TLR7 activation. c p53 expression in liver biopsy samples of chronic HBV patients compared to control steatosis patients. d Relative mRNA expression of different cell cycle regulators (p53, Cyclin E, Cyclin D1, Cyclin B1 and Cyclin A) on R837 treatment. e Detection of invitro cell proliferation on TLR7 activation using MTT assay. HepG2.2.15 cells were treated with R837 for 72 h and then incubated with MTT at the indicated time points
Figure Legend Snippet: Flow cytometric analysis and alteration of key cell cycle regulators upon TLR7 activation. HepG2.2.15 cells were treated with R837 (8μg/ml) for 72 h and then subjected to flow cytometric analysis to study the status of G1/S arrest. a Cell cycle analysis by FACS. b Percentage of cells in different phases of cell cycle before and after TLR7 activation. c p53 expression in liver biopsy samples of chronic HBV patients compared to control steatosis patients. d Relative mRNA expression of different cell cycle regulators (p53, Cyclin E, Cyclin D1, Cyclin B1 and Cyclin A) on R837 treatment. e Detection of invitro cell proliferation on TLR7 activation using MTT assay. HepG2.2.15 cells were treated with R837 for 72 h and then incubated with MTT at the indicated time points

Techniques Used: Flow Cytometry, Activation Assay, Cell Cycle Assay, FACS, Expressing, MTT Assay, Incubation

TLR7 pathway is activated on stimulation with its specific ligand imiquimod, R837. Chief downstream regulators, especially NF-ĸB is activated and transclocated to the nucleus on TLR7 activation. a Host immune response on TLR7 activation, using TLR7 ligand. HepG2.2.15 cells were treated with TLR7 agonist, R837 (8μg/ml) for 3 days. mRNA expression of different downstream regulators (NF- КB, JNK, p38 and PI3K) involved in the TLR7 pathway. b Confocal imaging showing nuclear uptake of Nuclear Factor-КB p65 subunit (NF- КB) from cytoplasm in cells where HepG2.2.15 cells are taken as control and HepG2.2.15 cells stimulated with TLR7 ligand, R837 (8μg/ml) for 72 h. c Nuclear Factor-КB p65 subunit (NF-КB) protein expression of HepG2 and HepG2.2.15 cells. d Nuclear Factor-КB p65 subunit (NF- КB) protein expression of HepG2.2.15 cells taken as control and treated with R837 (8μg/ml) for 72 h
Figure Legend Snippet: TLR7 pathway is activated on stimulation with its specific ligand imiquimod, R837. Chief downstream regulators, especially NF-ĸB is activated and transclocated to the nucleus on TLR7 activation. a Host immune response on TLR7 activation, using TLR7 ligand. HepG2.2.15 cells were treated with TLR7 agonist, R837 (8μg/ml) for 3 days. mRNA expression of different downstream regulators (NF- КB, JNK, p38 and PI3K) involved in the TLR7 pathway. b Confocal imaging showing nuclear uptake of Nuclear Factor-КB p65 subunit (NF- КB) from cytoplasm in cells where HepG2.2.15 cells are taken as control and HepG2.2.15 cells stimulated with TLR7 ligand, R837 (8μg/ml) for 72 h. c Nuclear Factor-КB p65 subunit (NF-КB) protein expression of HepG2 and HepG2.2.15 cells. d Nuclear Factor-КB p65 subunit (NF- КB) protein expression of HepG2.2.15 cells taken as control and treated with R837 (8μg/ml) for 72 h

Techniques Used: Activation Assay, Expressing, Imaging

TLR7 mediated cellular signaling pathway in suppression of HBV replication. HepG2.2.15 cells were pretreated with or without different pathway inhibitors (NF-КB pathway inhibitor PDTC, MAPK/JNK pathway inhibitor SP600125, PI3K-AKT inhibitor LY294002 and MAPK/p38 pathway inhibitor SB203580). After 1 h incubation, R837 was treated for 6 h. a Schematic representation of TLR7 signaling pathway and important inhibitors of relevant signaling molecules. b and c . HBV DNA, HBsAg AND HBeAg from culture supernatants on stimulation with R837 alone or pre-treated with different pathway inhibitors detected by real time RT- PCR and ELISA. d . Relative mRNA expression of JNK–dependent/ independent responsive genes (c-Jun, c-Myc, Elk-1 and SAP-1)
Figure Legend Snippet: TLR7 mediated cellular signaling pathway in suppression of HBV replication. HepG2.2.15 cells were pretreated with or without different pathway inhibitors (NF-КB pathway inhibitor PDTC, MAPK/JNK pathway inhibitor SP600125, PI3K-AKT inhibitor LY294002 and MAPK/p38 pathway inhibitor SB203580). After 1 h incubation, R837 was treated for 6 h. a Schematic representation of TLR7 signaling pathway and important inhibitors of relevant signaling molecules. b and c . HBV DNA, HBsAg AND HBeAg from culture supernatants on stimulation with R837 alone or pre-treated with different pathway inhibitors detected by real time RT- PCR and ELISA. d . Relative mRNA expression of JNK–dependent/ independent responsive genes (c-Jun, c-Myc, Elk-1 and SAP-1)

Techniques Used: Incubation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing

HBV titre was evaluated in culture supernatants of HepG2.2.15 cells after treatment with 4, 6 and 8μg/ml of TLR7 agonist (R837) for 72 h. a HBV DNA was isolated from the culture media of R837 treated cells and the load was assessed by absolute real-time PCR using WHO standards. b HBsAg and HBeAg were detected from the culture supernatant of treated cells by ELISA
Figure Legend Snippet: HBV titre was evaluated in culture supernatants of HepG2.2.15 cells after treatment with 4, 6 and 8μg/ml of TLR7 agonist (R837) for 72 h. a HBV DNA was isolated from the culture media of R837 treated cells and the load was assessed by absolute real-time PCR using WHO standards. b HBsAg and HBeAg were detected from the culture supernatant of treated cells by ELISA

Techniques Used: Isolation, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

4) Product Images from "Anti-hepatitis B virus (HBV) response of imiquimod based toll like receptor 7 ligand in hbv-positive human hepatocelluar carcinoma cell line"

Article Title: Anti-hepatitis B virus (HBV) response of imiquimod based toll like receptor 7 ligand in hbv-positive human hepatocelluar carcinoma cell line

Journal: BMC Infectious Diseases

doi: 10.1186/s12879-017-2189-z

Status of epigenetic markers and transcription factors on TLR7 activation. HepG2.2.15 cells were treated with R837 for 72 h and then the cells were subjected to Western Blot for protein expression studies. a Protein expression of different histone modifications (H3K4Me3, H3K9Me3, H3K36Me3 and H3K9Ac) and p53 before and after TLR7 activation. b The relative expression of the proteins (H3K9Me3 and p53) in HepG2 and HepG2.2.15 cells
Figure Legend Snippet: Status of epigenetic markers and transcription factors on TLR7 activation. HepG2.2.15 cells were treated with R837 for 72 h and then the cells were subjected to Western Blot for protein expression studies. a Protein expression of different histone modifications (H3K4Me3, H3K9Me3, H3K36Me3 and H3K9Ac) and p53 before and after TLR7 activation. b The relative expression of the proteins (H3K9Me3 and p53) in HepG2 and HepG2.2.15 cells

Techniques Used: Activation Assay, Western Blot, Expressing

Flow cytometric analysis and alteration of key cell cycle regulators upon TLR7 activation. HepG2.2.15 cells were treated with R837 (8μg/ml) for 72 h and then subjected to flow cytometric analysis to study the status of G1/S arrest. a Cell cycle analysis by FACS. b Percentage of cells in different phases of cell cycle before and after TLR7 activation. c p53 expression in liver biopsy samples of chronic HBV patients compared to control steatosis patients. d Relative mRNA expression of different cell cycle regulators (p53, Cyclin E, Cyclin D1, Cyclin B1 and Cyclin A) on R837 treatment. e Detection of invitro cell proliferation on TLR7 activation using MTT assay. HepG2.2.15 cells were treated with R837 for 72 h and then incubated with MTT at the indicated time points
Figure Legend Snippet: Flow cytometric analysis and alteration of key cell cycle regulators upon TLR7 activation. HepG2.2.15 cells were treated with R837 (8μg/ml) for 72 h and then subjected to flow cytometric analysis to study the status of G1/S arrest. a Cell cycle analysis by FACS. b Percentage of cells in different phases of cell cycle before and after TLR7 activation. c p53 expression in liver biopsy samples of chronic HBV patients compared to control steatosis patients. d Relative mRNA expression of different cell cycle regulators (p53, Cyclin E, Cyclin D1, Cyclin B1 and Cyclin A) on R837 treatment. e Detection of invitro cell proliferation on TLR7 activation using MTT assay. HepG2.2.15 cells were treated with R837 for 72 h and then incubated with MTT at the indicated time points

Techniques Used: Flow Cytometry, Activation Assay, Cell Cycle Assay, FACS, Expressing, MTT Assay, Incubation

TLR7 pathway is activated on stimulation with its specific ligand imiquimod, R837. Chief downstream regulators, especially NF-ĸB is activated and transclocated to the nucleus on TLR7 activation. a Host immune response on TLR7 activation, using TLR7 ligand. HepG2.2.15 cells were treated with TLR7 agonist, R837 (8μg/ml) for 3 days. mRNA expression of different downstream regulators (NF- КB, JNK, p38 and PI3K) involved in the TLR7 pathway. b Confocal imaging showing nuclear uptake of Nuclear Factor-КB p65 subunit (NF- КB) from cytoplasm in cells where HepG2.2.15 cells are taken as control and HepG2.2.15 cells stimulated with TLR7 ligand, R837 (8μg/ml) for 72 h. c Nuclear Factor-КB p65 subunit (NF-КB) protein expression of HepG2 and HepG2.2.15 cells. d Nuclear Factor-КB p65 subunit (NF- КB) protein expression of HepG2.2.15 cells taken as control and treated with R837 (8μg/ml) for 72 h
Figure Legend Snippet: TLR7 pathway is activated on stimulation with its specific ligand imiquimod, R837. Chief downstream regulators, especially NF-ĸB is activated and transclocated to the nucleus on TLR7 activation. a Host immune response on TLR7 activation, using TLR7 ligand. HepG2.2.15 cells were treated with TLR7 agonist, R837 (8μg/ml) for 3 days. mRNA expression of different downstream regulators (NF- КB, JNK, p38 and PI3K) involved in the TLR7 pathway. b Confocal imaging showing nuclear uptake of Nuclear Factor-КB p65 subunit (NF- КB) from cytoplasm in cells where HepG2.2.15 cells are taken as control and HepG2.2.15 cells stimulated with TLR7 ligand, R837 (8μg/ml) for 72 h. c Nuclear Factor-КB p65 subunit (NF-КB) protein expression of HepG2 and HepG2.2.15 cells. d Nuclear Factor-КB p65 subunit (NF- КB) protein expression of HepG2.2.15 cells taken as control and treated with R837 (8μg/ml) for 72 h

Techniques Used: Activation Assay, Expressing, Imaging

TLR7 mediated cellular signaling pathway in suppression of HBV replication. HepG2.2.15 cells were pretreated with or without different pathway inhibitors (NF-КB pathway inhibitor PDTC, MAPK/JNK pathway inhibitor SP600125, PI3K-AKT inhibitor LY294002 and MAPK/p38 pathway inhibitor SB203580). After 1 h incubation, R837 was treated for 6 h. a Schematic representation of TLR7 signaling pathway and important inhibitors of relevant signaling molecules. b and c . HBV DNA, HBsAg AND HBeAg from culture supernatants on stimulation with R837 alone or pre-treated with different pathway inhibitors detected by real time RT- PCR and ELISA. d . Relative mRNA expression of JNK–dependent/ independent responsive genes (c-Jun, c-Myc, Elk-1 and SAP-1)
Figure Legend Snippet: TLR7 mediated cellular signaling pathway in suppression of HBV replication. HepG2.2.15 cells were pretreated with or without different pathway inhibitors (NF-КB pathway inhibitor PDTC, MAPK/JNK pathway inhibitor SP600125, PI3K-AKT inhibitor LY294002 and MAPK/p38 pathway inhibitor SB203580). After 1 h incubation, R837 was treated for 6 h. a Schematic representation of TLR7 signaling pathway and important inhibitors of relevant signaling molecules. b and c . HBV DNA, HBsAg AND HBeAg from culture supernatants on stimulation with R837 alone or pre-treated with different pathway inhibitors detected by real time RT- PCR and ELISA. d . Relative mRNA expression of JNK–dependent/ independent responsive genes (c-Jun, c-Myc, Elk-1 and SAP-1)

Techniques Used: Incubation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing

HBV titre was evaluated in culture supernatants of HepG2.2.15 cells after treatment with 4, 6 and 8μg/ml of TLR7 agonist (R837) for 72 h. a HBV DNA was isolated from the culture media of R837 treated cells and the load was assessed by absolute real-time PCR using WHO standards. b HBsAg and HBeAg were detected from the culture supernatant of treated cells by ELISA
Figure Legend Snippet: HBV titre was evaluated in culture supernatants of HepG2.2.15 cells after treatment with 4, 6 and 8μg/ml of TLR7 agonist (R837) for 72 h. a HBV DNA was isolated from the culture media of R837 treated cells and the load was assessed by absolute real-time PCR using WHO standards. b HBsAg and HBeAg were detected from the culture supernatant of treated cells by ELISA

Techniques Used: Isolation, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

5) Product Images from "Anti-hepatitis B virus (HBV) response of imiquimod based toll like receptor 7 ligand in hbv-positive human hepatocelluar carcinoma cell line"

Article Title: Anti-hepatitis B virus (HBV) response of imiquimod based toll like receptor 7 ligand in hbv-positive human hepatocelluar carcinoma cell line

Journal: BMC Infectious Diseases

doi: 10.1186/s12879-017-2189-z

Status of epigenetic markers and transcription factors on TLR7 activation. HepG2.2.15 cells were treated with R837 for 72 h and then the cells were subjected to Western Blot for protein expression studies. a Protein expression of different histone modifications (H3K4Me3, H3K9Me3, H3K36Me3 and H3K9Ac) and p53 before and after TLR7 activation. b The relative expression of the proteins (H3K9Me3 and p53) in HepG2 and HepG2.2.15 cells
Figure Legend Snippet: Status of epigenetic markers and transcription factors on TLR7 activation. HepG2.2.15 cells were treated with R837 for 72 h and then the cells were subjected to Western Blot for protein expression studies. a Protein expression of different histone modifications (H3K4Me3, H3K9Me3, H3K36Me3 and H3K9Ac) and p53 before and after TLR7 activation. b The relative expression of the proteins (H3K9Me3 and p53) in HepG2 and HepG2.2.15 cells

Techniques Used: Activation Assay, Western Blot, Expressing

Flow cytometric analysis and alteration of key cell cycle regulators upon TLR7 activation. HepG2.2.15 cells were treated with R837 (8μg/ml) for 72 h and then subjected to flow cytometric analysis to study the status of G1/S arrest. a Cell cycle analysis by FACS. b Percentage of cells in different phases of cell cycle before and after TLR7 activation. c p53 expression in liver biopsy samples of chronic HBV patients compared to control steatosis patients. d Relative mRNA expression of different cell cycle regulators (p53, Cyclin E, Cyclin D1, Cyclin B1 and Cyclin A) on R837 treatment. e Detection of invitro cell proliferation on TLR7 activation using MTT assay. HepG2.2.15 cells were treated with R837 for 72 h and then incubated with MTT at the indicated time points
Figure Legend Snippet: Flow cytometric analysis and alteration of key cell cycle regulators upon TLR7 activation. HepG2.2.15 cells were treated with R837 (8μg/ml) for 72 h and then subjected to flow cytometric analysis to study the status of G1/S arrest. a Cell cycle analysis by FACS. b Percentage of cells in different phases of cell cycle before and after TLR7 activation. c p53 expression in liver biopsy samples of chronic HBV patients compared to control steatosis patients. d Relative mRNA expression of different cell cycle regulators (p53, Cyclin E, Cyclin D1, Cyclin B1 and Cyclin A) on R837 treatment. e Detection of invitro cell proliferation on TLR7 activation using MTT assay. HepG2.2.15 cells were treated with R837 for 72 h and then incubated with MTT at the indicated time points

Techniques Used: Flow Cytometry, Activation Assay, Cell Cycle Assay, FACS, Expressing, MTT Assay, Incubation

TLR7 pathway is activated on stimulation with its specific ligand imiquimod, R837. Chief downstream regulators, especially NF-ĸB is activated and transclocated to the nucleus on TLR7 activation. a Host immune response on TLR7 activation, using TLR7 ligand. HepG2.2.15 cells were treated with TLR7 agonist, R837 (8μg/ml) for 3 days. mRNA expression of different downstream regulators (NF- КB, JNK, p38 and PI3K) involved in the TLR7 pathway. b Confocal imaging showing nuclear uptake of Nuclear Factor-КB p65 subunit (NF- КB) from cytoplasm in cells where HepG2.2.15 cells are taken as control and HepG2.2.15 cells stimulated with TLR7 ligand, R837 (8μg/ml) for 72 h. c Nuclear Factor-КB p65 subunit (NF-КB) protein expression of HepG2 and HepG2.2.15 cells. d Nuclear Factor-КB p65 subunit (NF- КB) protein expression of HepG2.2.15 cells taken as control and treated with R837 (8μg/ml) for 72 h
Figure Legend Snippet: TLR7 pathway is activated on stimulation with its specific ligand imiquimod, R837. Chief downstream regulators, especially NF-ĸB is activated and transclocated to the nucleus on TLR7 activation. a Host immune response on TLR7 activation, using TLR7 ligand. HepG2.2.15 cells were treated with TLR7 agonist, R837 (8μg/ml) for 3 days. mRNA expression of different downstream regulators (NF- КB, JNK, p38 and PI3K) involved in the TLR7 pathway. b Confocal imaging showing nuclear uptake of Nuclear Factor-КB p65 subunit (NF- КB) from cytoplasm in cells where HepG2.2.15 cells are taken as control and HepG2.2.15 cells stimulated with TLR7 ligand, R837 (8μg/ml) for 72 h. c Nuclear Factor-КB p65 subunit (NF-КB) protein expression of HepG2 and HepG2.2.15 cells. d Nuclear Factor-КB p65 subunit (NF- КB) protein expression of HepG2.2.15 cells taken as control and treated with R837 (8μg/ml) for 72 h

Techniques Used: Activation Assay, Expressing, Imaging

TLR7 mediated cellular signaling pathway in suppression of HBV replication. HepG2.2.15 cells were pretreated with or without different pathway inhibitors (NF-КB pathway inhibitor PDTC, MAPK/JNK pathway inhibitor SP600125, PI3K-AKT inhibitor LY294002 and MAPK/p38 pathway inhibitor SB203580). After 1 h incubation, R837 was treated for 6 h. a Schematic representation of TLR7 signaling pathway and important inhibitors of relevant signaling molecules. b and c . HBV DNA, HBsAg AND HBeAg from culture supernatants on stimulation with R837 alone or pre-treated with different pathway inhibitors detected by real time RT- PCR and ELISA. d . Relative mRNA expression of JNK–dependent/ independent responsive genes (c-Jun, c-Myc, Elk-1 and SAP-1)
Figure Legend Snippet: TLR7 mediated cellular signaling pathway in suppression of HBV replication. HepG2.2.15 cells were pretreated with or without different pathway inhibitors (NF-КB pathway inhibitor PDTC, MAPK/JNK pathway inhibitor SP600125, PI3K-AKT inhibitor LY294002 and MAPK/p38 pathway inhibitor SB203580). After 1 h incubation, R837 was treated for 6 h. a Schematic representation of TLR7 signaling pathway and important inhibitors of relevant signaling molecules. b and c . HBV DNA, HBsAg AND HBeAg from culture supernatants on stimulation with R837 alone or pre-treated with different pathway inhibitors detected by real time RT- PCR and ELISA. d . Relative mRNA expression of JNK–dependent/ independent responsive genes (c-Jun, c-Myc, Elk-1 and SAP-1)

Techniques Used: Incubation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing

HBV titre was evaluated in culture supernatants of HepG2.2.15 cells after treatment with 4, 6 and 8μg/ml of TLR7 agonist (R837) for 72 h. a HBV DNA was isolated from the culture media of R837 treated cells and the load was assessed by absolute real-time PCR using WHO standards. b HBsAg and HBeAg were detected from the culture supernatant of treated cells by ELISA
Figure Legend Snippet: HBV titre was evaluated in culture supernatants of HepG2.2.15 cells after treatment with 4, 6 and 8μg/ml of TLR7 agonist (R837) for 72 h. a HBV DNA was isolated from the culture media of R837 treated cells and the load was assessed by absolute real-time PCR using WHO standards. b HBsAg and HBeAg were detected from the culture supernatant of treated cells by ELISA

Techniques Used: Isolation, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

6) Product Images from "Identification of a Novel Toll-like Receptor 7 Endogenous Ligand in Rheumatoid Arthritis Synovial Fluid That Can Provoke Arthritic Joint Inflammation"

Article Title: Identification of a Novel Toll-like Receptor 7 Endogenous Ligand in Rheumatoid Arthritis Synovial Fluid That Can Provoke Arthritic Joint Inflammation

Journal: Arthritis & rheumatology (Hoboken, N.J.)

doi: 10.1002/art.39544

Detection of accentuated microRNA let-7b (miR-let-7b) expression in rheumatoid arthritis (RA) synovial fluid (SF) macrophages. A, Copy numbers of miR-let-7b in normal (NL) plasma, RA plasma, osteoarthritis (OA) SF, and RA SF (n = 10–13 samples per group), as determined by real-time reverse transcription-polymerase chain reaction (RT-PCR). B and C, Exosomal secretion of miR-let-7b and miR-29a in peripheral blood (PB) T cells, in vitro-differentiated macrophages (Mac), and neutrophils (neutro) (n = 3–5 samples per group) ( B ) as well as in RA SF macrophages and RA synovial tissue (ST) fibroblasts (fibro) (n = 3 samples per group) ( C ). D and E, Copy numbers of miR-let-7b in RA SF macrophages ( D ) or RA ST ( E ). Samples (n = 3 per group) were left untreated (NT) or were treated for 6 hours with 1 µ M staurosporine to induce apoptosis or were subjected to repetitive freeze-thawing to promote necrosis. Exosomes released into the medium were extracted, and miR-let-7b copies were quantified. F, Levels of mRNA for tumor necrosis factor (TNF) and interleukin-6 (IL-6), as determined by real-time RT-PCR (n = 3 samples per group). RA PB in vitro-differentiated macrophages were left untreated (phosphate buffered saline [PBS] alone) or were treated for 6 hours with 5 µg/ml of miR-124, miR-147, or miR-let-7b. G and H, Western blot analysis of Toll-like receptor 7 (TLR-7) expression in control or knockdown cells and quantification of IL-6 ( G ) and IL-8 ( H ) transcription levels in in vitro-differentiated RA PB macrophages (n = 3 samples per group) after knockdown with small interfering RNA (siRNA). Cells were transfected for 72 hours with 100 n M control (Ctl-si) or TLR-7 (TLR7-si) siRNA and were left untreated or were stimulated for 6 hours with 1 µg/ml of R837 or with 5 µg/ml of miR-124, miR-147, or miR-let-7b. I, Western blot analysis and quantification of THP-1 cells transfected for 72 hours with 100 n M control, TLR-7, or TLR-8 siRNA Cells were left untreated or were stimulated for 6 hours with miR-let-7b, and IL-6 was quantified by real-time RT-PCR (n = 3 samples per group). The expression of TLRs 7 and 8 was markedly reduced after knockdown as compared to controls. Actin was used in Western blots as loading control. Fold change represents the increase over no treatment. Values are the mean ± SEM. * = P
Figure Legend Snippet: Detection of accentuated microRNA let-7b (miR-let-7b) expression in rheumatoid arthritis (RA) synovial fluid (SF) macrophages. A, Copy numbers of miR-let-7b in normal (NL) plasma, RA plasma, osteoarthritis (OA) SF, and RA SF (n = 10–13 samples per group), as determined by real-time reverse transcription-polymerase chain reaction (RT-PCR). B and C, Exosomal secretion of miR-let-7b and miR-29a in peripheral blood (PB) T cells, in vitro-differentiated macrophages (Mac), and neutrophils (neutro) (n = 3–5 samples per group) ( B ) as well as in RA SF macrophages and RA synovial tissue (ST) fibroblasts (fibro) (n = 3 samples per group) ( C ). D and E, Copy numbers of miR-let-7b in RA SF macrophages ( D ) or RA ST ( E ). Samples (n = 3 per group) were left untreated (NT) or were treated for 6 hours with 1 µ M staurosporine to induce apoptosis or were subjected to repetitive freeze-thawing to promote necrosis. Exosomes released into the medium were extracted, and miR-let-7b copies were quantified. F, Levels of mRNA for tumor necrosis factor (TNF) and interleukin-6 (IL-6), as determined by real-time RT-PCR (n = 3 samples per group). RA PB in vitro-differentiated macrophages were left untreated (phosphate buffered saline [PBS] alone) or were treated for 6 hours with 5 µg/ml of miR-124, miR-147, or miR-let-7b. G and H, Western blot analysis of Toll-like receptor 7 (TLR-7) expression in control or knockdown cells and quantification of IL-6 ( G ) and IL-8 ( H ) transcription levels in in vitro-differentiated RA PB macrophages (n = 3 samples per group) after knockdown with small interfering RNA (siRNA). Cells were transfected for 72 hours with 100 n M control (Ctl-si) or TLR-7 (TLR7-si) siRNA and were left untreated or were stimulated for 6 hours with 1 µg/ml of R837 or with 5 µg/ml of miR-124, miR-147, or miR-let-7b. I, Western blot analysis and quantification of THP-1 cells transfected for 72 hours with 100 n M control, TLR-7, or TLR-8 siRNA Cells were left untreated or were stimulated for 6 hours with miR-let-7b, and IL-6 was quantified by real-time RT-PCR (n = 3 samples per group). The expression of TLRs 7 and 8 was markedly reduced after knockdown as compared to controls. Actin was used in Western blots as loading control. Fold change represents the increase over no treatment. Values are the mean ± SEM. * = P

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, In Vitro, Quantitative RT-PCR, Western Blot, Small Interfering RNA, Transfection, CTL Assay

7) Product Images from "Identification of a Novel Toll-like Receptor 7 Endogenous Ligand in Rheumatoid Arthritis Synovial Fluid That Can Provoke Arthritic Joint Inflammation"

Article Title: Identification of a Novel Toll-like Receptor 7 Endogenous Ligand in Rheumatoid Arthritis Synovial Fluid That Can Provoke Arthritic Joint Inflammation

Journal: Arthritis & rheumatology (Hoboken, N.J.)

doi: 10.1002/art.39544

Detection of accentuated microRNA let-7b (miR-let-7b) expression in rheumatoid arthritis (RA) synovial fluid (SF) macrophages. A, Copy numbers of miR-let-7b in normal (NL) plasma, RA plasma, osteoarthritis (OA) SF, and RA SF (n = 10–13 samples per group), as determined by real-time reverse transcription-polymerase chain reaction (RT-PCR). B and C, Exosomal secretion of miR-let-7b and miR-29a in peripheral blood (PB) T cells, in vitro-differentiated macrophages (Mac), and neutrophils (neutro) (n = 3–5 samples per group) ( B ) as well as in RA SF macrophages and RA synovial tissue (ST) fibroblasts (fibro) (n = 3 samples per group) ( C ). D and E, Copy numbers of miR-let-7b in RA SF macrophages ( D ) or RA ST ( E ). Samples (n = 3 per group) were left untreated (NT) or were treated for 6 hours with 1 µ M staurosporine to induce apoptosis or were subjected to repetitive freeze-thawing to promote necrosis. Exosomes released into the medium were extracted, and miR-let-7b copies were quantified. F, Levels of mRNA for tumor necrosis factor (TNF) and interleukin-6 (IL-6), as determined by real-time RT-PCR (n = 3 samples per group). RA PB in vitro-differentiated macrophages were left untreated (phosphate buffered saline [PBS] alone) or were treated for 6 hours with 5 µg/ml of miR-124, miR-147, or miR-let-7b. G and H, Western blot analysis of Toll-like receptor 7 (TLR-7) expression in control or knockdown cells and quantification of IL-6 ( G ) and IL-8 ( H ) transcription levels in in vitro-differentiated RA PB macrophages (n = 3 samples per group) after knockdown with small interfering RNA (siRNA). Cells were transfected for 72 hours with 100 n M control (Ctl-si) or TLR-7 (TLR7-si) siRNA and were left untreated or were stimulated for 6 hours with 1 µg/ml of R837 or with 5 µg/ml of miR-124, miR-147, or miR-let-7b. I, Western blot analysis and quantification of THP-1 cells transfected for 72 hours with 100 n M control, TLR-7, or TLR-8 siRNA Cells were left untreated or were stimulated for 6 hours with miR-let-7b, and IL-6 was quantified by real-time RT-PCR (n = 3 samples per group). The expression of TLRs 7 and 8 was markedly reduced after knockdown as compared to controls. Actin was used in Western blots as loading control. Fold change represents the increase over no treatment. Values are the mean ± SEM. * = P
Figure Legend Snippet: Detection of accentuated microRNA let-7b (miR-let-7b) expression in rheumatoid arthritis (RA) synovial fluid (SF) macrophages. A, Copy numbers of miR-let-7b in normal (NL) plasma, RA plasma, osteoarthritis (OA) SF, and RA SF (n = 10–13 samples per group), as determined by real-time reverse transcription-polymerase chain reaction (RT-PCR). B and C, Exosomal secretion of miR-let-7b and miR-29a in peripheral blood (PB) T cells, in vitro-differentiated macrophages (Mac), and neutrophils (neutro) (n = 3–5 samples per group) ( B ) as well as in RA SF macrophages and RA synovial tissue (ST) fibroblasts (fibro) (n = 3 samples per group) ( C ). D and E, Copy numbers of miR-let-7b in RA SF macrophages ( D ) or RA ST ( E ). Samples (n = 3 per group) were left untreated (NT) or were treated for 6 hours with 1 µ M staurosporine to induce apoptosis or were subjected to repetitive freeze-thawing to promote necrosis. Exosomes released into the medium were extracted, and miR-let-7b copies were quantified. F, Levels of mRNA for tumor necrosis factor (TNF) and interleukin-6 (IL-6), as determined by real-time RT-PCR (n = 3 samples per group). RA PB in vitro-differentiated macrophages were left untreated (phosphate buffered saline [PBS] alone) or were treated for 6 hours with 5 µg/ml of miR-124, miR-147, or miR-let-7b. G and H, Western blot analysis of Toll-like receptor 7 (TLR-7) expression in control or knockdown cells and quantification of IL-6 ( G ) and IL-8 ( H ) transcription levels in in vitro-differentiated RA PB macrophages (n = 3 samples per group) after knockdown with small interfering RNA (siRNA). Cells were transfected for 72 hours with 100 n M control (Ctl-si) or TLR-7 (TLR7-si) siRNA and were left untreated or were stimulated for 6 hours with 1 µg/ml of R837 or with 5 µg/ml of miR-124, miR-147, or miR-let-7b. I, Western blot analysis and quantification of THP-1 cells transfected for 72 hours with 100 n M control, TLR-7, or TLR-8 siRNA Cells were left untreated or were stimulated for 6 hours with miR-let-7b, and IL-6 was quantified by real-time RT-PCR (n = 3 samples per group). The expression of TLRs 7 and 8 was markedly reduced after knockdown as compared to controls. Actin was used in Western blots as loading control. Fold change represents the increase over no treatment. Values are the mean ± SEM. * = P

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, In Vitro, Quantitative RT-PCR, Western Blot, Small Interfering RNA, Transfection, CTL Assay

8) Product Images from "Specific sequences of infectious challenge lead to secondary hemophagocytic lymphohistiocytosis-like disease in mice"

Article Title: Specific sequences of infectious challenge lead to secondary hemophagocytic lymphohistiocytosis-like disease in mice

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1820704116

Viral TLR agonism primes a hyperinflammatory LPS response. ( A ) Survival of 8-wk-old male C57Bl6/J mice treated with 5 mg/kg LPS i.p. or 10 mg/kg i.v. Poly I:C at the indicated times (arrows) in the indicated order ( n = 10 mice per group). ( B ) Colonic temperatures of 8-wk-old male C57Bl6/J mice treated with LPS:IC or IC:LPS ( n = 10 per group). ( C ) Survival of 8-wk-old male C57Bl6/J mice treated with 10 mg/kg Poly I:C, 5 mg/kg LPS, 10 mg/kg R837 i.p., 10 mg/kg CpG ODN1826 i.p. in the indicated order at the indicated times (arrows) ( n > 8 per group). ( D ) Survival of 8-wk-old male C57Bl6/J, caspase 1/11 DKO, IFNaR KO, TLR2/4 DKO, or MyD88/TRIF DKO mice were challenged with IC:LPS ( n > 8 per group). ( E – H ) Plasma IL-6, TNFα, IL-1β, or IFNα assessed following LPS and Poly I:C administered at the indicated time points (arrows) in the indicated order ( n > 5 per group). **** P
Figure Legend Snippet: Viral TLR agonism primes a hyperinflammatory LPS response. ( A ) Survival of 8-wk-old male C57Bl6/J mice treated with 5 mg/kg LPS i.p. or 10 mg/kg i.v. Poly I:C at the indicated times (arrows) in the indicated order ( n = 10 mice per group). ( B ) Colonic temperatures of 8-wk-old male C57Bl6/J mice treated with LPS:IC or IC:LPS ( n = 10 per group). ( C ) Survival of 8-wk-old male C57Bl6/J mice treated with 10 mg/kg Poly I:C, 5 mg/kg LPS, 10 mg/kg R837 i.p., 10 mg/kg CpG ODN1826 i.p. in the indicated order at the indicated times (arrows) ( n > 8 per group). ( D ) Survival of 8-wk-old male C57Bl6/J, caspase 1/11 DKO, IFNaR KO, TLR2/4 DKO, or MyD88/TRIF DKO mice were challenged with IC:LPS ( n > 8 per group). ( E – H ) Plasma IL-6, TNFα, IL-1β, or IFNα assessed following LPS and Poly I:C administered at the indicated time points (arrows) in the indicated order ( n > 5 per group). **** P

Techniques Used: Mouse Assay

9) Product Images from "Anti-hepatitis B virus (HBV) response of imiquimod based toll like receptor 7 ligand in hbv-positive human hepatocelluar carcinoma cell line"

Article Title: Anti-hepatitis B virus (HBV) response of imiquimod based toll like receptor 7 ligand in hbv-positive human hepatocelluar carcinoma cell line

Journal: BMC Infectious Diseases

doi: 10.1186/s12879-017-2189-z

Status of epigenetic markers and transcription factors on TLR7 activation. HepG2.2.15 cells were treated with R837 for 72 h and then the cells were subjected to Western Blot for protein expression studies. a Protein expression of different histone modifications (H3K4Me3, H3K9Me3, H3K36Me3 and H3K9Ac) and p53 before and after TLR7 activation. b The relative expression of the proteins (H3K9Me3 and p53) in HepG2 and HepG2.2.15 cells
Figure Legend Snippet: Status of epigenetic markers and transcription factors on TLR7 activation. HepG2.2.15 cells were treated with R837 for 72 h and then the cells were subjected to Western Blot for protein expression studies. a Protein expression of different histone modifications (H3K4Me3, H3K9Me3, H3K36Me3 and H3K9Ac) and p53 before and after TLR7 activation. b The relative expression of the proteins (H3K9Me3 and p53) in HepG2 and HepG2.2.15 cells

Techniques Used: Activation Assay, Western Blot, Expressing

Flow cytometric analysis and alteration of key cell cycle regulators upon TLR7 activation. HepG2.2.15 cells were treated with R837 (8μg/ml) for 72 h and then subjected to flow cytometric analysis to study the status of G1/S arrest. a Cell cycle analysis by FACS. b Percentage of cells in different phases of cell cycle before and after TLR7 activation. c p53 expression in liver biopsy samples of chronic HBV patients compared to control steatosis patients. d Relative mRNA expression of different cell cycle regulators (p53, Cyclin E, Cyclin D1, Cyclin B1 and Cyclin A) on R837 treatment. e Detection of invitro cell proliferation on TLR7 activation using MTT assay. HepG2.2.15 cells were treated with R837 for 72 h and then incubated with MTT at the indicated time points
Figure Legend Snippet: Flow cytometric analysis and alteration of key cell cycle regulators upon TLR7 activation. HepG2.2.15 cells were treated with R837 (8μg/ml) for 72 h and then subjected to flow cytometric analysis to study the status of G1/S arrest. a Cell cycle analysis by FACS. b Percentage of cells in different phases of cell cycle before and after TLR7 activation. c p53 expression in liver biopsy samples of chronic HBV patients compared to control steatosis patients. d Relative mRNA expression of different cell cycle regulators (p53, Cyclin E, Cyclin D1, Cyclin B1 and Cyclin A) on R837 treatment. e Detection of invitro cell proliferation on TLR7 activation using MTT assay. HepG2.2.15 cells were treated with R837 for 72 h and then incubated with MTT at the indicated time points

Techniques Used: Flow Cytometry, Activation Assay, Cell Cycle Assay, FACS, Expressing, MTT Assay, Incubation

TLR7 pathway is activated on stimulation with its specific ligand imiquimod, R837. Chief downstream regulators, especially NF-ĸB is activated and transclocated to the nucleus on TLR7 activation. a Host immune response on TLR7 activation, using TLR7 ligand. HepG2.2.15 cells were treated with TLR7 agonist, R837 (8μg/ml) for 3 days. mRNA expression of different downstream regulators (NF- КB, JNK, p38 and PI3K) involved in the TLR7 pathway. b Confocal imaging showing nuclear uptake of Nuclear Factor-КB p65 subunit (NF- КB) from cytoplasm in cells where HepG2.2.15 cells are taken as control and HepG2.2.15 cells stimulated with TLR7 ligand, R837 (8μg/ml) for 72 h. c Nuclear Factor-КB p65 subunit (NF-КB) protein expression of HepG2 and HepG2.2.15 cells. d Nuclear Factor-КB p65 subunit (NF- КB) protein expression of HepG2.2.15 cells taken as control and treated with R837 (8μg/ml) for 72 h
Figure Legend Snippet: TLR7 pathway is activated on stimulation with its specific ligand imiquimod, R837. Chief downstream regulators, especially NF-ĸB is activated and transclocated to the nucleus on TLR7 activation. a Host immune response on TLR7 activation, using TLR7 ligand. HepG2.2.15 cells were treated with TLR7 agonist, R837 (8μg/ml) for 3 days. mRNA expression of different downstream regulators (NF- КB, JNK, p38 and PI3K) involved in the TLR7 pathway. b Confocal imaging showing nuclear uptake of Nuclear Factor-КB p65 subunit (NF- КB) from cytoplasm in cells where HepG2.2.15 cells are taken as control and HepG2.2.15 cells stimulated with TLR7 ligand, R837 (8μg/ml) for 72 h. c Nuclear Factor-КB p65 subunit (NF-КB) protein expression of HepG2 and HepG2.2.15 cells. d Nuclear Factor-КB p65 subunit (NF- КB) protein expression of HepG2.2.15 cells taken as control and treated with R837 (8μg/ml) for 72 h

Techniques Used: Activation Assay, Expressing, Imaging

TLR7 mediated cellular signaling pathway in suppression of HBV replication. HepG2.2.15 cells were pretreated with or without different pathway inhibitors (NF-КB pathway inhibitor PDTC, MAPK/JNK pathway inhibitor SP600125, PI3K-AKT inhibitor LY294002 and MAPK/p38 pathway inhibitor SB203580). After 1 h incubation, R837 was treated for 6 h. a Schematic representation of TLR7 signaling pathway and important inhibitors of relevant signaling molecules. b and c . HBV DNA, HBsAg AND HBeAg from culture supernatants on stimulation with R837 alone or pre-treated with different pathway inhibitors detected by real time RT- PCR and ELISA. d . Relative mRNA expression of JNK–dependent/ independent responsive genes (c-Jun, c-Myc, Elk-1 and SAP-1)
Figure Legend Snippet: TLR7 mediated cellular signaling pathway in suppression of HBV replication. HepG2.2.15 cells were pretreated with or without different pathway inhibitors (NF-КB pathway inhibitor PDTC, MAPK/JNK pathway inhibitor SP600125, PI3K-AKT inhibitor LY294002 and MAPK/p38 pathway inhibitor SB203580). After 1 h incubation, R837 was treated for 6 h. a Schematic representation of TLR7 signaling pathway and important inhibitors of relevant signaling molecules. b and c . HBV DNA, HBsAg AND HBeAg from culture supernatants on stimulation with R837 alone or pre-treated with different pathway inhibitors detected by real time RT- PCR and ELISA. d . Relative mRNA expression of JNK–dependent/ independent responsive genes (c-Jun, c-Myc, Elk-1 and SAP-1)

Techniques Used: Incubation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing

HBV titre was evaluated in culture supernatants of HepG2.2.15 cells after treatment with 4, 6 and 8μg/ml of TLR7 agonist (R837) for 72 h. a HBV DNA was isolated from the culture media of R837 treated cells and the load was assessed by absolute real-time PCR using WHO standards. b HBsAg and HBeAg were detected from the culture supernatant of treated cells by ELISA
Figure Legend Snippet: HBV titre was evaluated in culture supernatants of HepG2.2.15 cells after treatment with 4, 6 and 8μg/ml of TLR7 agonist (R837) for 72 h. a HBV DNA was isolated from the culture media of R837 treated cells and the load was assessed by absolute real-time PCR using WHO standards. b HBsAg and HBeAg were detected from the culture supernatant of treated cells by ELISA

Techniques Used: Isolation, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

10) Product Images from "Blimp-1-Mediated Pathway Promotes Type I IFN Production in Plasmacytoid Dendritic Cells by Targeting to Interleukin-1 Receptor-Associated Kinase M"

Article Title: Blimp-1-Mediated Pathway Promotes Type I IFN Production in Plasmacytoid Dendritic Cells by Targeting to Interleukin-1 Receptor-Associated Kinase M

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01828

Blimp-1 was induced via Rac activation after TLR7/9 ligand treatment in plasmacytoid dendritic cells (pDCs). (A) RT-quantitative PCR (RT-qPCR) showing Blimp-1 mRNA in human pDCs 24 h after treatment with medium alone, 1 µM CpG-A and H1N1 at a titer of 10 4 TCID50/ml. (B) Blimp-1 protein levels were determined by immunoblotting in human pDCs 24 h after treatment with medium alone or 1 µM CpG-A. (C) ELISA showing the levels of IFN-α produced by human pDCs as described in (A) . (D) Blimp-1-yellow fluorescent protein reporter mice were intravenously injected with DOTAP alone or DOTAP + CpG-A. The frequency of Blimp-1 + pDCs in splenic CD11c int B220 + Siglec-H + Bst2 + gate was examined at indicated time after infection. The frequency of Blimp-1 + pDCs from untreated group (naïve) was shown for comparison. (E) Nuclear Blimp-1 protein levels were detected by immunoblotting in mouse FLpDCs stimulated with medium alone or 1 µM CpG-A at indicated time points. Freshly isolated FLpDCs at 0 h, before addition of medium alone or CpG-A, were also used as the control. (F) RT-qPCR showing the Blimp-1 mRNA levels in Tlr9 knockout (KO) FLpDCs treated with 1 µM CpG-A. (G) RT-qPCR showing Blimp-1 mRNA levels in FLpDCs after 1 h pre-treatment with EHop-016 and further treatment with 1 µM CpG-A for 1 h. (H) RT-qPCR showing the Blimp-1 mRNA levels in Tlr7 KO FLpDCs treated with 2 µg/ml R837 for 1 h. (I) Rac1 activation determined by PAK1 PBD agarose beads pulled down and immunoblotting with antibody against Rac1 in FLpDCs from WT and TLR7 KO mice after stimulation with 2 µg/ml R837. (J) RT-qPCR showing Blimp-1 mRNA expression in FLpDCs after 1 h pre-treatment with EHop-016 and further treatment with 2 µg/ml R837 for 1 h. Data represent the mean ± SEM and were analyzed by two-tailed unpaired Student’s t -test [ n = 3−6 in (A) , 4−7 in (C) , 3 in (F) , 5−6 in (G) , 3 in (H) , and 4−5 in (J) ]. * p
Figure Legend Snippet: Blimp-1 was induced via Rac activation after TLR7/9 ligand treatment in plasmacytoid dendritic cells (pDCs). (A) RT-quantitative PCR (RT-qPCR) showing Blimp-1 mRNA in human pDCs 24 h after treatment with medium alone, 1 µM CpG-A and H1N1 at a titer of 10 4 TCID50/ml. (B) Blimp-1 protein levels were determined by immunoblotting in human pDCs 24 h after treatment with medium alone or 1 µM CpG-A. (C) ELISA showing the levels of IFN-α produced by human pDCs as described in (A) . (D) Blimp-1-yellow fluorescent protein reporter mice were intravenously injected with DOTAP alone or DOTAP + CpG-A. The frequency of Blimp-1 + pDCs in splenic CD11c int B220 + Siglec-H + Bst2 + gate was examined at indicated time after infection. The frequency of Blimp-1 + pDCs from untreated group (naïve) was shown for comparison. (E) Nuclear Blimp-1 protein levels were detected by immunoblotting in mouse FLpDCs stimulated with medium alone or 1 µM CpG-A at indicated time points. Freshly isolated FLpDCs at 0 h, before addition of medium alone or CpG-A, were also used as the control. (F) RT-qPCR showing the Blimp-1 mRNA levels in Tlr9 knockout (KO) FLpDCs treated with 1 µM CpG-A. (G) RT-qPCR showing Blimp-1 mRNA levels in FLpDCs after 1 h pre-treatment with EHop-016 and further treatment with 1 µM CpG-A for 1 h. (H) RT-qPCR showing the Blimp-1 mRNA levels in Tlr7 KO FLpDCs treated with 2 µg/ml R837 for 1 h. (I) Rac1 activation determined by PAK1 PBD agarose beads pulled down and immunoblotting with antibody against Rac1 in FLpDCs from WT and TLR7 KO mice after stimulation with 2 µg/ml R837. (J) RT-qPCR showing Blimp-1 mRNA expression in FLpDCs after 1 h pre-treatment with EHop-016 and further treatment with 2 µg/ml R837 for 1 h. Data represent the mean ± SEM and were analyzed by two-tailed unpaired Student’s t -test [ n = 3−6 in (A) , 4−7 in (C) , 3 in (F) , 5−6 in (G) , 3 in (H) , and 4−5 in (J) ]. * p

Techniques Used: Activation Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Produced, Mouse Assay, Injection, Infection, Isolation, Knock-Out, Expressing, Two Tailed Test

11) Product Images from "Hypoxia and therapeutic treatment of EV-A71 with an immune modulator TLR7 agonist in a new immunocompetent mouse model"

Article Title: Hypoxia and therapeutic treatment of EV-A71 with an immune modulator TLR7 agonist in a new immunocompetent mouse model

Journal: Journal of Biomedical Science

doi: 10.1186/s12929-019-0585-y

TLR7 agonist R837 rescued mice from paralysis and death efficiently after viral infection. Upper panel: A schematic diagram of R837 treatment via an oral or i.p. route after EV-A71 infection. Lower panel: Mice were treated with or without R837 (5.0 μg /g of mouse) at dpi = 1. Significant reduction in clinical score was observed only in the two groups treated with R837. *** p
Figure Legend Snippet: TLR7 agonist R837 rescued mice from paralysis and death efficiently after viral infection. Upper panel: A schematic diagram of R837 treatment via an oral or i.p. route after EV-A71 infection. Lower panel: Mice were treated with or without R837 (5.0 μg /g of mouse) at dpi = 1. Significant reduction in clinical score was observed only in the two groups treated with R837. *** p

Techniques Used: Mouse Assay, Infection

12) Product Images from "TSG-6 Downregulates IFN-Alpha and TNF-Alpha Expression by Suppressing IRF7 Phosphorylation in Human Plasmacytoid Dendritic Cells"

Article Title: TSG-6 Downregulates IFN-Alpha and TNF-Alpha Expression by Suppressing IRF7 Phosphorylation in Human Plasmacytoid Dendritic Cells

Journal: Mediators of Inflammation

doi: 10.1155/2017/7462945

CpG-A and R837 induced TSG-6 expression in GEN2.2 cells. (a and b) Real-time PCR analysis and (c) ELISA analysis of GEN2.2 cells treated with CpG-A (2 μ M) or R837 (10 ng/mL) shows induction of TSG-6 at both transcription and protein level. Graphs show data of at least 3 independent experiments performed in duplicate. Error bars indicate SEM. Paired Student's t -test, ∗ p
Figure Legend Snippet: CpG-A and R837 induced TSG-6 expression in GEN2.2 cells. (a and b) Real-time PCR analysis and (c) ELISA analysis of GEN2.2 cells treated with CpG-A (2 μ M) or R837 (10 ng/mL) shows induction of TSG-6 at both transcription and protein level. Graphs show data of at least 3 independent experiments performed in duplicate. Error bars indicate SEM. Paired Student's t -test, ∗ p

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

TSG-6 increased CD44 expression. Real-time PCR (a and b) and flow-cytometric analysis (c and d) of GEN2.2 cells treated with TSG-6 (1000 ng/mL), followed by stimulation with CpG-A (2 μ M) or R837 (10 ng/mL) for 3 hours. Representative FACS plots showing CD44+ pDCs percentages per independent experiment ( n > 3). Graphs showing data of at least 3 experiments performed in duplicate. Error bars indicate SEM. One-Way ANOVA, ∗ p
Figure Legend Snippet: TSG-6 increased CD44 expression. Real-time PCR (a and b) and flow-cytometric analysis (c and d) of GEN2.2 cells treated with TSG-6 (1000 ng/mL), followed by stimulation with CpG-A (2 μ M) or R837 (10 ng/mL) for 3 hours. Representative FACS plots showing CD44+ pDCs percentages per independent experiment ( n > 3). Graphs showing data of at least 3 experiments performed in duplicate. Error bars indicate SEM. One-Way ANOVA, ∗ p

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, FACS

TSG-6 reduced the transcription of interferon regulatory factor 7 and its associated genes in GEN2.2. TSG-6 reduced the transcription of interferon regulatory factor 7 and its associated genes in GEN2.2. Real-time PCR analysis of IRF7, IFN- α 2, TNF- α , and IL-1 β in GEN2.2 cells treated with TSG-6 (1000 ng/mL) overnight, followed by stimulation with CpG-A at 2 μ M (a) or R837 at 10 ng/mL (b) for 3 hours. Graphs show data of more than 3 experiments performed in duplicate. Error bars indicate SEM. One-Way ANOVA, ∗ p
Figure Legend Snippet: TSG-6 reduced the transcription of interferon regulatory factor 7 and its associated genes in GEN2.2. TSG-6 reduced the transcription of interferon regulatory factor 7 and its associated genes in GEN2.2. Real-time PCR analysis of IRF7, IFN- α 2, TNF- α , and IL-1 β in GEN2.2 cells treated with TSG-6 (1000 ng/mL) overnight, followed by stimulation with CpG-A at 2 μ M (a) or R837 at 10 ng/mL (b) for 3 hours. Graphs show data of more than 3 experiments performed in duplicate. Error bars indicate SEM. One-Way ANOVA, ∗ p

Techniques Used: Real-time Polymerase Chain Reaction

TSG-6 exerted the same suppressive effect on pDCs from healthy donors stimulated with CpG-A or R837. PBMC from 14 healthy blood donors were isolated and treated with TSG-6 (1000 ng/mL) overnight, followed by CpG-A (2 μ M) (a) or R837 (10 ng/mL) (b) for 5 hours. The cells were stained with the cell surface markers as indicated, fixed, and permeabilized, followed by staining with APC-conjugated anti-IFN- α and FITC-conjugated anti-TNF- α antibodies. PBMCs were gated for pDCs as defined in the gating strategy (Supplementary data S2). MFI, mean fluorescence intensity. Error bars indicate SEM. Wilcoxon matched-pairs signed rank test was used to determine statistical significance. ∗∗ p
Figure Legend Snippet: TSG-6 exerted the same suppressive effect on pDCs from healthy donors stimulated with CpG-A or R837. PBMC from 14 healthy blood donors were isolated and treated with TSG-6 (1000 ng/mL) overnight, followed by CpG-A (2 μ M) (a) or R837 (10 ng/mL) (b) for 5 hours. The cells were stained with the cell surface markers as indicated, fixed, and permeabilized, followed by staining with APC-conjugated anti-IFN- α and FITC-conjugated anti-TNF- α antibodies. PBMCs were gated for pDCs as defined in the gating strategy (Supplementary data S2). MFI, mean fluorescence intensity. Error bars indicate SEM. Wilcoxon matched-pairs signed rank test was used to determine statistical significance. ∗∗ p

Techniques Used: Isolation, Staining, Fluorescence

TSG-6 reduced the activation of IRF7 in GEN2.2 cells. GEN2.2 cells were treated with TSG-6 (1000 ng/mL) overnight, followed by stimulation with CpG-A (2 μ M) (a) or R837 (10 ng/mL) (b) for 30 min. The cells were then fixed, permeabilized, and stained to detect phosphorylated IRF7 (phosphor-IRF7) at Ser477 and Ser479 by flow cytometry. The bar chart shows the means of 5 independent experiments, indicating the IRF7 phosphorylation activity in GEN2.2 cells. Error bars indicate SEM. Paired Student's t -test, ∗∗ p
Figure Legend Snippet: TSG-6 reduced the activation of IRF7 in GEN2.2 cells. GEN2.2 cells were treated with TSG-6 (1000 ng/mL) overnight, followed by stimulation with CpG-A (2 μ M) (a) or R837 (10 ng/mL) (b) for 30 min. The cells were then fixed, permeabilized, and stained to detect phosphorylated IRF7 (phosphor-IRF7) at Ser477 and Ser479 by flow cytometry. The bar chart shows the means of 5 independent experiments, indicating the IRF7 phosphorylation activity in GEN2.2 cells. Error bars indicate SEM. Paired Student's t -test, ∗∗ p

Techniques Used: Activation Assay, Staining, Flow Cytometry, Cytometry, Activity Assay

TSG-6 decreased TLR7/9 mediated TNF- α and IFN- α production in GEN2.2 cells. ELISA analysis of TNF- α (a) and IFN- α (b) in GEN2.2 cells treated with TSG-6 (1000 ng/mL) overnight, with or without stimulation with CpG-A at 2 μ M or R837 at 10 ng/mL for 6 hours. Graphs show data from three independent experiments performed in duplicate. Error bars indicate SEM. Paired Student's t -test, ∗ p
Figure Legend Snippet: TSG-6 decreased TLR7/9 mediated TNF- α and IFN- α production in GEN2.2 cells. ELISA analysis of TNF- α (a) and IFN- α (b) in GEN2.2 cells treated with TSG-6 (1000 ng/mL) overnight, with or without stimulation with CpG-A at 2 μ M or R837 at 10 ng/mL for 6 hours. Graphs show data from three independent experiments performed in duplicate. Error bars indicate SEM. Paired Student's t -test, ∗ p

Techniques Used: Enzyme-linked Immunosorbent Assay

The suppressive effect of TSG-6 was dependent on CD44. GEN 2.2 cells were treated with anti-CD44 blocking antibody at various concentrations as indicated or with control IgG (mouse IgG2b) for 45 min, followed by TSG-6 (1000 ng/mL) overnight, and stimulation with CpG-A (2 μ M) (a) or R837 (10 ng/mL) (b) for 6 hours. Culture supernatants were collected for TNF- α and IFN- α quantification by ELISA. Graphs showing data from three independent experiments performed in duplicate. Error bars indicate SEM. One-Way ANOVA, ∗ p
Figure Legend Snippet: The suppressive effect of TSG-6 was dependent on CD44. GEN 2.2 cells were treated with anti-CD44 blocking antibody at various concentrations as indicated or with control IgG (mouse IgG2b) for 45 min, followed by TSG-6 (1000 ng/mL) overnight, and stimulation with CpG-A (2 μ M) (a) or R837 (10 ng/mL) (b) for 6 hours. Culture supernatants were collected for TNF- α and IFN- α quantification by ELISA. Graphs showing data from three independent experiments performed in duplicate. Error bars indicate SEM. One-Way ANOVA, ∗ p

Techniques Used: Blocking Assay, Enzyme-linked Immunosorbent Assay

13) Product Images from "Wiskott-Aldrich syndrome protein-mediated actin dynamics control type-I interferon production in plasmacytoid dendritic cells"

Article Title: Wiskott-Aldrich syndrome protein-mediated actin dynamics control type-I interferon production in plasmacytoid dendritic cells

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20120363

Progressive exhaustion of the TLR9/IFN-α in WKO mice. (A) WT and WKO adult ( > 6 wk) mice were injected intravenously with 10 µg CpG-A in complex with the cationic lipid DOTAP. Serum levels of IFN-α, IL-6, and IL-12p40 were measured by ELISA at 2, 6, 10, and 24 h after injection. n = 4–9 mice per group in three independent experiments. (B) Dot plots showing IFN-α intracellular staining in the spleen of WT and WKO mice isolated at different time points after injection. Numbers represents the percentage of cells in the respective quadrant. Data are representative of one of three experiments performed each with three mice per group. (C) Gene transcription and protein release in splenic pDCs sorted from WT and WKO adult mice. Cells were stimulated ex vivo with CpG-A, CpG-B, and R837 (15 µg/ml). The relative expression of Ifna4 transcripts was evaluated by qRT–PCR at 4 and 24 h. The target mRNA was normalized to β-actin mRNA. Values are shown as the 2 ΔCT × 10 3 . The levels of IFN-α (C, right), IL-6, and IL-12p40 (D) were measured in cell culture supernatants after 16 h. Results are representative of four (CpG-A and CpG-B in C) and three (R837 in C and D) independent experiments. A, Mann-Whitney test; C and D, Student’s t test; error bars indicate SEM. *, P
Figure Legend Snippet: Progressive exhaustion of the TLR9/IFN-α in WKO mice. (A) WT and WKO adult ( > 6 wk) mice were injected intravenously with 10 µg CpG-A in complex with the cationic lipid DOTAP. Serum levels of IFN-α, IL-6, and IL-12p40 were measured by ELISA at 2, 6, 10, and 24 h after injection. n = 4–9 mice per group in three independent experiments. (B) Dot plots showing IFN-α intracellular staining in the spleen of WT and WKO mice isolated at different time points after injection. Numbers represents the percentage of cells in the respective quadrant. Data are representative of one of three experiments performed each with three mice per group. (C) Gene transcription and protein release in splenic pDCs sorted from WT and WKO adult mice. Cells were stimulated ex vivo with CpG-A, CpG-B, and R837 (15 µg/ml). The relative expression of Ifna4 transcripts was evaluated by qRT–PCR at 4 and 24 h. The target mRNA was normalized to β-actin mRNA. Values are shown as the 2 ΔCT × 10 3 . The levels of IFN-α (C, right), IL-6, and IL-12p40 (D) were measured in cell culture supernatants after 16 h. Results are representative of four (CpG-A and CpG-B in C) and three (R837 in C and D) independent experiments. A, Mann-Whitney test; C and D, Student’s t test; error bars indicate SEM. *, P

Techniques Used: Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay, Staining, Isolation, Ex Vivo, Expressing, Quantitative RT-PCR, Cell Culture, MANN-WHITNEY

Cell-intrinsic role of WASp-mediated actin dynamics in limiting IFN - α production in pDCs. (A) Responses to TLR9 and TLR7 agonists in naive pDCs from young WKO mice. 3 × 10 5 pDCs isolated from the spleen of young (3 wk) WT or WKO mice were stimulated ex vivo with 10 µg/ml CpG-A or CpG-B and 5 µg/ml R837. Bars show IFN-α, IL-6, and TNF protein levels determined by ELISA in cell culture supernatant. Results are from two (R837) to five (CpG-A and CpG-B) independent experiments each with six mice per group. (B) pDCs were stimulated with 20 µg/ml CpG-A for 5 h in the presence of brefeldin A. Intracellular staining was performed with antibodies against IFN-α and TNF. The plots are from one representative experiment of three performed. (C) Splenic pDCs from young WT or WKO mice were incubated with sera from WT ( n = 4) or WKO ( n = 4) adult mice. Data show the levels of IFN-α in cell culture supernatants subtracted of the values in control wells without cells. Data are representative of two experiments with six mice per group. (D and E) BM-pDCs or BM-cDCs were nucleofected with siRNA control (si-irr) or WASp-specific (si-WASp) RNA oligos. (D) 3 × 10 5 control or WASp-silenced BM-pDCs were stimulated with 15 µg/ml CpG-A or CpG-B. IFN-α and IL-6 protein levels were determined by ELISA. Results are from four experiments. (E) 3 × 10 5 control or WASp-silenced BM-cDCs were stimulated with 15 µg/ml CpG-B. IL-12p40 and IL-6 protein levels were determined by ELISA. Results are representative of three experiments. (F) 3 × 10 5 BM-pDCs were preincubated with 1 µg/ml cytochalasin- d (Cyto- d ), 0.1 µg/ml latrunculin-A or control medium, washed and stimulated with 5 µg/ml CpG-A for 5 h. Bars show concentrations of IFN-α and IL-6 protein in cell culture supernatants. Data are representative of two (latrunculin-A) to six (cytochalasin- d ) independent experiments. Levels of IL-6 (G) and IL-12p40 (H) induced by CpG-B in Cyto-D–treated pDCs and Cyto-D–treated cDCs upon stimulation with 15 µg/ml CpG-A and CpG-B, respectively. One representative experiment out of three with similar results is shown. A, D, and E, Student’s t test; F, Mann-Whitney test; error bars indicate SEM. *, P
Figure Legend Snippet: Cell-intrinsic role of WASp-mediated actin dynamics in limiting IFN - α production in pDCs. (A) Responses to TLR9 and TLR7 agonists in naive pDCs from young WKO mice. 3 × 10 5 pDCs isolated from the spleen of young (3 wk) WT or WKO mice were stimulated ex vivo with 10 µg/ml CpG-A or CpG-B and 5 µg/ml R837. Bars show IFN-α, IL-6, and TNF protein levels determined by ELISA in cell culture supernatant. Results are from two (R837) to five (CpG-A and CpG-B) independent experiments each with six mice per group. (B) pDCs were stimulated with 20 µg/ml CpG-A for 5 h in the presence of brefeldin A. Intracellular staining was performed with antibodies against IFN-α and TNF. The plots are from one representative experiment of three performed. (C) Splenic pDCs from young WT or WKO mice were incubated with sera from WT ( n = 4) or WKO ( n = 4) adult mice. Data show the levels of IFN-α in cell culture supernatants subtracted of the values in control wells without cells. Data are representative of two experiments with six mice per group. (D and E) BM-pDCs or BM-cDCs were nucleofected with siRNA control (si-irr) or WASp-specific (si-WASp) RNA oligos. (D) 3 × 10 5 control or WASp-silenced BM-pDCs were stimulated with 15 µg/ml CpG-A or CpG-B. IFN-α and IL-6 protein levels were determined by ELISA. Results are from four experiments. (E) 3 × 10 5 control or WASp-silenced BM-cDCs were stimulated with 15 µg/ml CpG-B. IL-12p40 and IL-6 protein levels were determined by ELISA. Results are representative of three experiments. (F) 3 × 10 5 BM-pDCs were preincubated with 1 µg/ml cytochalasin- d (Cyto- d ), 0.1 µg/ml latrunculin-A or control medium, washed and stimulated with 5 µg/ml CpG-A for 5 h. Bars show concentrations of IFN-α and IL-6 protein in cell culture supernatants. Data are representative of two (latrunculin-A) to six (cytochalasin- d ) independent experiments. Levels of IL-6 (G) and IL-12p40 (H) induced by CpG-B in Cyto-D–treated pDCs and Cyto-D–treated cDCs upon stimulation with 15 µg/ml CpG-A and CpG-B, respectively. One representative experiment out of three with similar results is shown. A, D, and E, Student’s t test; F, Mann-Whitney test; error bars indicate SEM. *, P

Techniques Used: Mouse Assay, Isolation, Ex Vivo, Enzyme-linked Immunosorbent Assay, Cell Culture, Staining, Incubation, MANN-WHITNEY

14) Product Images from "Anti-hepatitis B virus (HBV) response of imiquimod based toll like receptor 7 ligand in hbv-positive human hepatocelluar carcinoma cell line"

Article Title: Anti-hepatitis B virus (HBV) response of imiquimod based toll like receptor 7 ligand in hbv-positive human hepatocelluar carcinoma cell line

Journal: BMC Infectious Diseases

doi: 10.1186/s12879-017-2189-z

Status of epigenetic markers and transcription factors on TLR7 activation. HepG2.2.15 cells were treated with R837 for 72 h and then the cells were subjected to Western Blot for protein expression studies. a Protein expression of different histone modifications (H3K4Me3, H3K9Me3, H3K36Me3 and H3K9Ac) and p53 before and after TLR7 activation. b The relative expression of the proteins (H3K9Me3 and p53) in HepG2 and HepG2.2.15 cells
Figure Legend Snippet: Status of epigenetic markers and transcription factors on TLR7 activation. HepG2.2.15 cells were treated with R837 for 72 h and then the cells were subjected to Western Blot for protein expression studies. a Protein expression of different histone modifications (H3K4Me3, H3K9Me3, H3K36Me3 and H3K9Ac) and p53 before and after TLR7 activation. b The relative expression of the proteins (H3K9Me3 and p53) in HepG2 and HepG2.2.15 cells

Techniques Used: Activation Assay, Western Blot, Expressing

Flow cytometric analysis and alteration of key cell cycle regulators upon TLR7 activation. HepG2.2.15 cells were treated with R837 (8μg/ml) for 72 h and then subjected to flow cytometric analysis to study the status of G1/S arrest. a Cell cycle analysis by FACS. b Percentage of cells in different phases of cell cycle before and after TLR7 activation. c p53 expression in liver biopsy samples of chronic HBV patients compared to control steatosis patients. d Relative mRNA expression of different cell cycle regulators (p53, Cyclin E, Cyclin D1, Cyclin B1 and Cyclin A) on R837 treatment. e Detection of invitro cell proliferation on TLR7 activation using MTT assay. HepG2.2.15 cells were treated with R837 for 72 h and then incubated with MTT at the indicated time points
Figure Legend Snippet: Flow cytometric analysis and alteration of key cell cycle regulators upon TLR7 activation. HepG2.2.15 cells were treated with R837 (8μg/ml) for 72 h and then subjected to flow cytometric analysis to study the status of G1/S arrest. a Cell cycle analysis by FACS. b Percentage of cells in different phases of cell cycle before and after TLR7 activation. c p53 expression in liver biopsy samples of chronic HBV patients compared to control steatosis patients. d Relative mRNA expression of different cell cycle regulators (p53, Cyclin E, Cyclin D1, Cyclin B1 and Cyclin A) on R837 treatment. e Detection of invitro cell proliferation on TLR7 activation using MTT assay. HepG2.2.15 cells were treated with R837 for 72 h and then incubated with MTT at the indicated time points

Techniques Used: Flow Cytometry, Activation Assay, Cell Cycle Assay, FACS, Expressing, MTT Assay, Incubation

TLR7 pathway is activated on stimulation with its specific ligand imiquimod, R837. Chief downstream regulators, especially NF-ĸB is activated and transclocated to the nucleus on TLR7 activation. a Host immune response on TLR7 activation, using TLR7 ligand. HepG2.2.15 cells were treated with TLR7 agonist, R837 (8μg/ml) for 3 days. mRNA expression of different downstream regulators (NF- КB, JNK, p38 and PI3K) involved in the TLR7 pathway. b Confocal imaging showing nuclear uptake of Nuclear Factor-КB p65 subunit (NF- КB) from cytoplasm in cells where HepG2.2.15 cells are taken as control and HepG2.2.15 cells stimulated with TLR7 ligand, R837 (8μg/ml) for 72 h. c Nuclear Factor-КB p65 subunit (NF-КB) protein expression of HepG2 and HepG2.2.15 cells. d Nuclear Factor-КB p65 subunit (NF- КB) protein expression of HepG2.2.15 cells taken as control and treated with R837 (8μg/ml) for 72 h
Figure Legend Snippet: TLR7 pathway is activated on stimulation with its specific ligand imiquimod, R837. Chief downstream regulators, especially NF-ĸB is activated and transclocated to the nucleus on TLR7 activation. a Host immune response on TLR7 activation, using TLR7 ligand. HepG2.2.15 cells were treated with TLR7 agonist, R837 (8μg/ml) for 3 days. mRNA expression of different downstream regulators (NF- КB, JNK, p38 and PI3K) involved in the TLR7 pathway. b Confocal imaging showing nuclear uptake of Nuclear Factor-КB p65 subunit (NF- КB) from cytoplasm in cells where HepG2.2.15 cells are taken as control and HepG2.2.15 cells stimulated with TLR7 ligand, R837 (8μg/ml) for 72 h. c Nuclear Factor-КB p65 subunit (NF-КB) protein expression of HepG2 and HepG2.2.15 cells. d Nuclear Factor-КB p65 subunit (NF- КB) protein expression of HepG2.2.15 cells taken as control and treated with R837 (8μg/ml) for 72 h

Techniques Used: Activation Assay, Expressing, Imaging

TLR7 mediated cellular signaling pathway in suppression of HBV replication. HepG2.2.15 cells were pretreated with or without different pathway inhibitors (NF-КB pathway inhibitor PDTC, MAPK/JNK pathway inhibitor SP600125, PI3K-AKT inhibitor LY294002 and MAPK/p38 pathway inhibitor SB203580). After 1 h incubation, R837 was treated for 6 h. a Schematic representation of TLR7 signaling pathway and important inhibitors of relevant signaling molecules. b and c . HBV DNA, HBsAg AND HBeAg from culture supernatants on stimulation with R837 alone or pre-treated with different pathway inhibitors detected by real time RT- PCR and ELISA. d . Relative mRNA expression of JNK–dependent/ independent responsive genes (c-Jun, c-Myc, Elk-1 and SAP-1)
Figure Legend Snippet: TLR7 mediated cellular signaling pathway in suppression of HBV replication. HepG2.2.15 cells were pretreated with or without different pathway inhibitors (NF-КB pathway inhibitor PDTC, MAPK/JNK pathway inhibitor SP600125, PI3K-AKT inhibitor LY294002 and MAPK/p38 pathway inhibitor SB203580). After 1 h incubation, R837 was treated for 6 h. a Schematic representation of TLR7 signaling pathway and important inhibitors of relevant signaling molecules. b and c . HBV DNA, HBsAg AND HBeAg from culture supernatants on stimulation with R837 alone or pre-treated with different pathway inhibitors detected by real time RT- PCR and ELISA. d . Relative mRNA expression of JNK–dependent/ independent responsive genes (c-Jun, c-Myc, Elk-1 and SAP-1)

Techniques Used: Incubation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing

HBV titre was evaluated in culture supernatants of HepG2.2.15 cells after treatment with 4, 6 and 8μg/ml of TLR7 agonist (R837) for 72 h. a HBV DNA was isolated from the culture media of R837 treated cells and the load was assessed by absolute real-time PCR using WHO standards. b HBsAg and HBeAg were detected from the culture supernatant of treated cells by ELISA
Figure Legend Snippet: HBV titre was evaluated in culture supernatants of HepG2.2.15 cells after treatment with 4, 6 and 8μg/ml of TLR7 agonist (R837) for 72 h. a HBV DNA was isolated from the culture media of R837 treated cells and the load was assessed by absolute real-time PCR using WHO standards. b HBsAg and HBeAg were detected from the culture supernatant of treated cells by ELISA

Techniques Used: Isolation, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

15) Product Images from "Inverse Immunological Responses Induced by Allergic Rhinitis and Head and Neck Squamous Cell Carcinoma"

Article Title: Inverse Immunological Responses Induced by Allergic Rhinitis and Head and Neck Squamous Cell Carcinoma

Journal: PLoS ONE

doi: 10.1371/journal.pone.0086796

Decreased peripheral blood mononuclear cells (PBMC) activation in patients with head and neck squamous cell carcinoma (HNSCC). PBMC were isolated from blood collected from healthy controls (n = 10), patients with an ongoing seasonal allergic rhinitis (AR; n = 11) and patients with HNSCC (n = 9), and cultured with or without Pam 3 CSK 4 (1 µg/ml), LPS (1 µg/ml), R837 (5 µg/ml) or CpG (0.3 µM) for 24 h. ( A ) Thereafter, the supernatants were analyzed for the secreted cytokine profile with Luminex Multiplex Immunoassay, 4 out of 17 investigated cytokines are shown, ( B ) and the cells were examined for the expression of CD25 and CD98 on CD4 positive T helper (Th) cells with flow cytometry. The results are presented as the non-stimulated value minus the TLR stimulated values. Grey colored samples were analyzed on a BD LSRFortessa, whereas the rest of the samples were investigated on a Beckman Coulter Navios flow cytometer. One representative histogram from the healthy controls, AR patients and HNSCC patients is displayed. The open histograms represent LPS stimulated cells, and the light grey histograms are denoting the non-stimulated samples. ( C ) RNA was extracted from peripheral PBMC, isolated from healthy individuals (n = 8), AR patients (n = 10), and HNSCC patients (n = 7), made into cDNA, and thereafter analyzed for COX-1 and COX-2 mRNA expression with real-time PCR. Data are given in relation to the housekeeping gene β-actin, 100 000×2 −ΔCt , and presented as mean ± SEM. MFI = mean fluorescence intensity; * p ≤0.05; ** p ≤0.01; *** p ≤0.001.
Figure Legend Snippet: Decreased peripheral blood mononuclear cells (PBMC) activation in patients with head and neck squamous cell carcinoma (HNSCC). PBMC were isolated from blood collected from healthy controls (n = 10), patients with an ongoing seasonal allergic rhinitis (AR; n = 11) and patients with HNSCC (n = 9), and cultured with or without Pam 3 CSK 4 (1 µg/ml), LPS (1 µg/ml), R837 (5 µg/ml) or CpG (0.3 µM) for 24 h. ( A ) Thereafter, the supernatants were analyzed for the secreted cytokine profile with Luminex Multiplex Immunoassay, 4 out of 17 investigated cytokines are shown, ( B ) and the cells were examined for the expression of CD25 and CD98 on CD4 positive T helper (Th) cells with flow cytometry. The results are presented as the non-stimulated value minus the TLR stimulated values. Grey colored samples were analyzed on a BD LSRFortessa, whereas the rest of the samples were investigated on a Beckman Coulter Navios flow cytometer. One representative histogram from the healthy controls, AR patients and HNSCC patients is displayed. The open histograms represent LPS stimulated cells, and the light grey histograms are denoting the non-stimulated samples. ( C ) RNA was extracted from peripheral PBMC, isolated from healthy individuals (n = 8), AR patients (n = 10), and HNSCC patients (n = 7), made into cDNA, and thereafter analyzed for COX-1 and COX-2 mRNA expression with real-time PCR. Data are given in relation to the housekeeping gene β-actin, 100 000×2 −ΔCt , and presented as mean ± SEM. MFI = mean fluorescence intensity; * p ≤0.05; ** p ≤0.01; *** p ≤0.001.

Techniques Used: Activation Assay, Isolation, Cell Culture, Luminex, Multiplex Assay, Expressing, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Fluorescence

Increased activation of polymorphonuclear leukocytes (PMN) in patients with head and neck squamous cell carcinoma (HNSCC). Blood was obtained from healthy controls (n = 10), patients with an ongoing seasonal allergic rhinitis (AR; n = 11) and patients with HNSCC (ELISA n = 9; FACS n = 10). PMN were isolated and cultured in the presence or absence of Pam 3 CSK 4 (1 µg/ml), LPS (1 µg/ml), R837 (5 µg/ml) or CpG (0.3 µM) for 4 and 24 h. ( A ) The cell free supernatants were then analyzed for IL-6 and IL-8 with ELISA, ( B ) and the CD16 positive cells were investigated for the expression of CD11b and CD69 using flow cytometry. The results are presented as the non-stimulated value minus the TLR stimulated values. Grey colored samples were analyzed on a BD LSRFortessa, whereas the rest of the samples were investigated on a Beckman Coulter Navios flow cytometer. ( C ) PMN from healthy individuals (n = 9) were incubated with or without culture medium supernatant from the HNSCC cell line FaDu, and stimulated with or without 1 µg/ml LPS for 4 h, and analyzed for IL-6 and IL-8 secretion with ELISA. The basal cytokine levels in the media were subtracted from the concentrations present in the PMN cultures. MFI = mean fluorescence intensity; * p ≤0.05; ** p ≤0.01; *** p ≤0.001.
Figure Legend Snippet: Increased activation of polymorphonuclear leukocytes (PMN) in patients with head and neck squamous cell carcinoma (HNSCC). Blood was obtained from healthy controls (n = 10), patients with an ongoing seasonal allergic rhinitis (AR; n = 11) and patients with HNSCC (ELISA n = 9; FACS n = 10). PMN were isolated and cultured in the presence or absence of Pam 3 CSK 4 (1 µg/ml), LPS (1 µg/ml), R837 (5 µg/ml) or CpG (0.3 µM) for 4 and 24 h. ( A ) The cell free supernatants were then analyzed for IL-6 and IL-8 with ELISA, ( B ) and the CD16 positive cells were investigated for the expression of CD11b and CD69 using flow cytometry. The results are presented as the non-stimulated value minus the TLR stimulated values. Grey colored samples were analyzed on a BD LSRFortessa, whereas the rest of the samples were investigated on a Beckman Coulter Navios flow cytometer. ( C ) PMN from healthy individuals (n = 9) were incubated with or without culture medium supernatant from the HNSCC cell line FaDu, and stimulated with or without 1 µg/ml LPS for 4 h, and analyzed for IL-6 and IL-8 secretion with ELISA. The basal cytokine levels in the media were subtracted from the concentrations present in the PMN cultures. MFI = mean fluorescence intensity; * p ≤0.05; ** p ≤0.01; *** p ≤0.001.

Techniques Used: Activation Assay, Enzyme-linked Immunosorbent Assay, FACS, Isolation, Cell Culture, Expressing, Flow Cytometry, Cytometry, Incubation, Fluorescence

16) Product Images from "Identification of a Novel Toll-like Receptor 7 Endogenous Ligand in Rheumatoid Arthritis Synovial Fluid That Can Provoke Arthritic Joint Inflammation"

Article Title: Identification of a Novel Toll-like Receptor 7 Endogenous Ligand in Rheumatoid Arthritis Synovial Fluid That Can Provoke Arthritic Joint Inflammation

Journal: Arthritis & rheumatology (Hoboken, N.J.)

doi: 10.1002/art.39544

Detection of accentuated microRNA let-7b (miR-let-7b) expression in rheumatoid arthritis (RA) synovial fluid (SF) macrophages. A, Copy numbers of miR-let-7b in normal (NL) plasma, RA plasma, osteoarthritis (OA) SF, and RA SF (n = 10–13 samples per group), as determined by real-time reverse transcription-polymerase chain reaction (RT-PCR). B and C, Exosomal secretion of miR-let-7b and miR-29a in peripheral blood (PB) T cells, in vitro-differentiated macrophages (Mac), and neutrophils (neutro) (n = 3–5 samples per group) ( B ) as well as in RA SF macrophages and RA synovial tissue (ST) fibroblasts (fibro) (n = 3 samples per group) ( C ). D and E, Copy numbers of miR-let-7b in RA SF macrophages ( D ) or RA ST ( E ). Samples (n = 3 per group) were left untreated (NT) or were treated for 6 hours with 1 µ M staurosporine to induce apoptosis or were subjected to repetitive freeze-thawing to promote necrosis. Exosomes released into the medium were extracted, and miR-let-7b copies were quantified. F, Levels of mRNA for tumor necrosis factor (TNF) and interleukin-6 (IL-6), as determined by real-time RT-PCR (n = 3 samples per group). RA PB in vitro-differentiated macrophages were left untreated (phosphate buffered saline [PBS] alone) or were treated for 6 hours with 5 µg/ml of miR-124, miR-147, or miR-let-7b. G and H, Western blot analysis of Toll-like receptor 7 (TLR-7) expression in control or knockdown cells and quantification of IL-6 ( G ) and IL-8 ( H ) transcription levels in in vitro-differentiated RA PB macrophages (n = 3 samples per group) after knockdown with small interfering RNA (siRNA). Cells were transfected for 72 hours with 100 n M control (Ctl-si) or TLR-7 (TLR7-si) siRNA and were left untreated or were stimulated for 6 hours with 1 µg/ml of R837 or with 5 µg/ml of miR-124, miR-147, or miR-let-7b. I, Western blot analysis and quantification of THP-1 cells transfected for 72 hours with 100 n M control, TLR-7, or TLR-8 siRNA Cells were left untreated or were stimulated for 6 hours with miR-let-7b, and IL-6 was quantified by real-time RT-PCR (n = 3 samples per group). The expression of TLRs 7 and 8 was markedly reduced after knockdown as compared to controls. Actin was used in Western blots as loading control. Fold change represents the increase over no treatment. Values are the mean ± SEM. * = P
Figure Legend Snippet: Detection of accentuated microRNA let-7b (miR-let-7b) expression in rheumatoid arthritis (RA) synovial fluid (SF) macrophages. A, Copy numbers of miR-let-7b in normal (NL) plasma, RA plasma, osteoarthritis (OA) SF, and RA SF (n = 10–13 samples per group), as determined by real-time reverse transcription-polymerase chain reaction (RT-PCR). B and C, Exosomal secretion of miR-let-7b and miR-29a in peripheral blood (PB) T cells, in vitro-differentiated macrophages (Mac), and neutrophils (neutro) (n = 3–5 samples per group) ( B ) as well as in RA SF macrophages and RA synovial tissue (ST) fibroblasts (fibro) (n = 3 samples per group) ( C ). D and E, Copy numbers of miR-let-7b in RA SF macrophages ( D ) or RA ST ( E ). Samples (n = 3 per group) were left untreated (NT) or were treated for 6 hours with 1 µ M staurosporine to induce apoptosis or were subjected to repetitive freeze-thawing to promote necrosis. Exosomes released into the medium were extracted, and miR-let-7b copies were quantified. F, Levels of mRNA for tumor necrosis factor (TNF) and interleukin-6 (IL-6), as determined by real-time RT-PCR (n = 3 samples per group). RA PB in vitro-differentiated macrophages were left untreated (phosphate buffered saline [PBS] alone) or were treated for 6 hours with 5 µg/ml of miR-124, miR-147, or miR-let-7b. G and H, Western blot analysis of Toll-like receptor 7 (TLR-7) expression in control or knockdown cells and quantification of IL-6 ( G ) and IL-8 ( H ) transcription levels in in vitro-differentiated RA PB macrophages (n = 3 samples per group) after knockdown with small interfering RNA (siRNA). Cells were transfected for 72 hours with 100 n M control (Ctl-si) or TLR-7 (TLR7-si) siRNA and were left untreated or were stimulated for 6 hours with 1 µg/ml of R837 or with 5 µg/ml of miR-124, miR-147, or miR-let-7b. I, Western blot analysis and quantification of THP-1 cells transfected for 72 hours with 100 n M control, TLR-7, or TLR-8 siRNA Cells were left untreated or were stimulated for 6 hours with miR-let-7b, and IL-6 was quantified by real-time RT-PCR (n = 3 samples per group). The expression of TLRs 7 and 8 was markedly reduced after knockdown as compared to controls. Actin was used in Western blots as loading control. Fold change represents the increase over no treatment. Values are the mean ± SEM. * = P

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, In Vitro, Quantitative RT-PCR, Western Blot, Small Interfering RNA, Transfection, CTL Assay

17) Product Images from "Transcription Factor ATF4 Induces NLRP1 Inflammasome Expression during Endoplasmic Reticulum Stress"

Article Title: Transcription Factor ATF4 Induces NLRP1 Inflammasome Expression during Endoplasmic Reticulum Stress

Journal: PLoS ONE

doi: 10.1371/journal.pone.0130635

NLRP1 mRNA and protein are up-regulated upon ER stress. (A) Un-differentiated THP-1 cells were treated with the indicated stimuli for 6 hours. NLRP1 levels were measured by quantitative real-time PCR (qPCR) using cyclophillin A as an endogenous control. Semi-quantitative RT-PCR using a different NLRP1 primer set and GAPDH as a control is also shown. (B) HeLa cells were treated either with BFA or TG for the indicated times. NLRP1 mRNA levels were measured by qPCR and RT-PCR. Spliced and un-spliced XBP-1 forms were also evaluated by RT-PCR. (C) HCT116 cells were treated with the indicated stimuli for 24 hours. NLRP1 and NOD1 mRNA levels were measured by qPCR. (D) Cell lysates from wild-type or NLRP1 −/− HeLa, THP-1 and K562 cells, untreated or treated with BFA for 20 hours, were normalized for total protein content. Cell extracts were then subjected to SDS-PAGE/immunoblot analysis before and after immunoprecipitation with NLRP1 antibody. Vinculin was detected as loading control. NLRP1 mRNA levels were also measured by RT-PCR. Each panel is representative of at least three independent experiments. (DMSO: dimethyl sulfoxide, TM: tunicamycin, TG: thapsigargin, MSU: monosodium urate crystals, BFA: brefeldin A, PolyI:C: polyinosinic-polycytidylic acid, FLA: flagellin, MDP: muramyl dipeptide, R837: Imiquimod)
Figure Legend Snippet: NLRP1 mRNA and protein are up-regulated upon ER stress. (A) Un-differentiated THP-1 cells were treated with the indicated stimuli for 6 hours. NLRP1 levels were measured by quantitative real-time PCR (qPCR) using cyclophillin A as an endogenous control. Semi-quantitative RT-PCR using a different NLRP1 primer set and GAPDH as a control is also shown. (B) HeLa cells were treated either with BFA or TG for the indicated times. NLRP1 mRNA levels were measured by qPCR and RT-PCR. Spliced and un-spliced XBP-1 forms were also evaluated by RT-PCR. (C) HCT116 cells were treated with the indicated stimuli for 24 hours. NLRP1 and NOD1 mRNA levels were measured by qPCR. (D) Cell lysates from wild-type or NLRP1 −/− HeLa, THP-1 and K562 cells, untreated or treated with BFA for 20 hours, were normalized for total protein content. Cell extracts were then subjected to SDS-PAGE/immunoblot analysis before and after immunoprecipitation with NLRP1 antibody. Vinculin was detected as loading control. NLRP1 mRNA levels were also measured by RT-PCR. Each panel is representative of at least three independent experiments. (DMSO: dimethyl sulfoxide, TM: tunicamycin, TG: thapsigargin, MSU: monosodium urate crystals, BFA: brefeldin A, PolyI:C: polyinosinic-polycytidylic acid, FLA: flagellin, MDP: muramyl dipeptide, R837: Imiquimod)

Techniques Used: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, SDS Page, Immunoprecipitation

18) Product Images from "Circulating Plasma Extracellular Vesicles from Septic Mice Induce Inflammation via MicroRNA- and TLR7-Dependent Mechanisms"

Article Title: Circulating Plasma Extracellular Vesicles from Septic Mice Induce Inflammation via MicroRNA- and TLR7-Dependent Mechanisms

Journal: The Journal of Immunology Author Choice

doi: 10.4049/jimmunol.1801008

Plasma EVs from septic mice induce cytokine production through TLR7-MyD88 signaling. BMDM were isolated from WT and genetically modified mice (TLR7 −/− , TLR3 −/− , MyD88 −/− , Trif −/− ) and treated with sham EVs (20 μg/ml), CLP EVs (20 μg/ml), P3C (TLR2 ligand, 1 μg/ml), poly(I:C) (TLR3 ligands, 10 μg/ml), or R837 (TLR7 ligand, 0.25 μg/ml). Sixteen hours after the treatment, the culture media were collected and cytokines (MIP-2, IL-6) were measured by ELISA. ( A and B ) TLR7 but not TLR3 signaling contributed to CLP EV–induced cytokine production. ( C and D ) MyD88- but not Trif-deficiency abolished CLP EV–mediated MIP-2 and IL-6 production. Each bar represents triplicate samples with each experiment repeated twice. *** p
Figure Legend Snippet: Plasma EVs from septic mice induce cytokine production through TLR7-MyD88 signaling. BMDM were isolated from WT and genetically modified mice (TLR7 −/− , TLR3 −/− , MyD88 −/− , Trif −/− ) and treated with sham EVs (20 μg/ml), CLP EVs (20 μg/ml), P3C (TLR2 ligand, 1 μg/ml), poly(I:C) (TLR3 ligands, 10 μg/ml), or R837 (TLR7 ligand, 0.25 μg/ml). Sixteen hours after the treatment, the culture media were collected and cytokines (MIP-2, IL-6) were measured by ELISA. ( A and B ) TLR7 but not TLR3 signaling contributed to CLP EV–induced cytokine production. ( C and D ) MyD88- but not Trif-deficiency abolished CLP EV–mediated MIP-2 and IL-6 production. Each bar represents triplicate samples with each experiment repeated twice. *** p

Techniques Used: Mouse Assay, Isolation, Genetically Modified, Enzyme-linked Immunosorbent Assay

19) Product Images from "Wiskott-Aldrich syndrome protein-mediated actin dynamics control type-I interferon production in plasmacytoid dendritic cells"

Article Title: Wiskott-Aldrich syndrome protein-mediated actin dynamics control type-I interferon production in plasmacytoid dendritic cells

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20120363

Progressive exhaustion of the TLR9/IFN-α in WKO mice. (A) WT and WKO adult ( > 6 wk) mice were injected intravenously with 10 µg CpG-A in complex with the cationic lipid DOTAP. Serum levels of IFN-α, IL-6, and IL-12p40 were measured by ELISA at 2, 6, 10, and 24 h after injection. n = 4–9 mice per group in three independent experiments. (B) Dot plots showing IFN-α intracellular staining in the spleen of WT and WKO mice isolated at different time points after injection. Numbers represents the percentage of cells in the respective quadrant. Data are representative of one of three experiments performed each with three mice per group. (C) Gene transcription and protein release in splenic pDCs sorted from WT and WKO adult mice. Cells were stimulated ex vivo with CpG-A, CpG-B, and R837 (15 µg/ml). The relative expression of Ifna4 transcripts was evaluated by qRT–PCR at 4 and 24 h. The target mRNA was normalized to β-actin mRNA. Values are shown as the 2 ΔCT × 10 3 . The levels of IFN-α (C, right), IL-6, and IL-12p40 (D) were measured in cell culture supernatants after 16 h. Results are representative of four (CpG-A and CpG-B in C) and three (R837 in C and D) independent experiments. A, Mann-Whitney test; C and D, Student’s t test; error bars indicate SEM. *, P
Figure Legend Snippet: Progressive exhaustion of the TLR9/IFN-α in WKO mice. (A) WT and WKO adult ( > 6 wk) mice were injected intravenously with 10 µg CpG-A in complex with the cationic lipid DOTAP. Serum levels of IFN-α, IL-6, and IL-12p40 were measured by ELISA at 2, 6, 10, and 24 h after injection. n = 4–9 mice per group in three independent experiments. (B) Dot plots showing IFN-α intracellular staining in the spleen of WT and WKO mice isolated at different time points after injection. Numbers represents the percentage of cells in the respective quadrant. Data are representative of one of three experiments performed each with three mice per group. (C) Gene transcription and protein release in splenic pDCs sorted from WT and WKO adult mice. Cells were stimulated ex vivo with CpG-A, CpG-B, and R837 (15 µg/ml). The relative expression of Ifna4 transcripts was evaluated by qRT–PCR at 4 and 24 h. The target mRNA was normalized to β-actin mRNA. Values are shown as the 2 ΔCT × 10 3 . The levels of IFN-α (C, right), IL-6, and IL-12p40 (D) were measured in cell culture supernatants after 16 h. Results are representative of four (CpG-A and CpG-B in C) and three (R837 in C and D) independent experiments. A, Mann-Whitney test; C and D, Student’s t test; error bars indicate SEM. *, P

Techniques Used: Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay, Staining, Isolation, Ex Vivo, Expressing, Quantitative RT-PCR, Cell Culture, MANN-WHITNEY

Cell-intrinsic role of WASp-mediated actin dynamics in limiting IFN - α production in pDCs. (A) Responses to TLR9 and TLR7 agonists in naive pDCs from young WKO mice. 3 × 10 5 pDCs isolated from the spleen of young (3 wk) WT or WKO mice were stimulated ex vivo with 10 µg/ml CpG-A or CpG-B and 5 µg/ml R837. Bars show IFN-α, IL-6, and TNF protein levels determined by ELISA in cell culture supernatant. Results are from two (R837) to five (CpG-A and CpG-B) independent experiments each with six mice per group. (B) pDCs were stimulated with 20 µg/ml CpG-A for 5 h in the presence of brefeldin A. Intracellular staining was performed with antibodies against IFN-α and TNF. The plots are from one representative experiment of three performed. (C) Splenic pDCs from young WT or WKO mice were incubated with sera from WT ( n = 4) or WKO ( n = 4) adult mice. Data show the levels of IFN-α in cell culture supernatants subtracted of the values in control wells without cells. Data are representative of two experiments with six mice per group. (D and E) BM-pDCs or BM-cDCs were nucleofected with siRNA control (si-irr) or WASp-specific (si-WASp) RNA oligos. (D) 3 × 10 5 control or WASp-silenced BM-pDCs were stimulated with 15 µg/ml CpG-A or CpG-B. IFN-α and IL-6 protein levels were determined by ELISA. Results are from four experiments. (E) 3 × 10 5 control or WASp-silenced BM-cDCs were stimulated with 15 µg/ml CpG-B. IL-12p40 and IL-6 protein levels were determined by ELISA. Results are representative of three experiments. (F) 3 × 10 5 BM-pDCs were preincubated with 1 µg/ml cytochalasin- d (Cyto- d ), 0.1 µg/ml latrunculin-A or control medium, washed and stimulated with 5 µg/ml CpG-A for 5 h. Bars show concentrations of IFN-α and IL-6 protein in cell culture supernatants. Data are representative of two (latrunculin-A) to six (cytochalasin- d ) independent experiments. Levels of IL-6 (G) and IL-12p40 (H) induced by CpG-B in Cyto-D–treated pDCs and Cyto-D–treated cDCs upon stimulation with 15 µg/ml CpG-A and CpG-B, respectively. One representative experiment out of three with similar results is shown. A, D, and E, Student’s t test; F, Mann-Whitney test; error bars indicate SEM. *, P
Figure Legend Snippet: Cell-intrinsic role of WASp-mediated actin dynamics in limiting IFN - α production in pDCs. (A) Responses to TLR9 and TLR7 agonists in naive pDCs from young WKO mice. 3 × 10 5 pDCs isolated from the spleen of young (3 wk) WT or WKO mice were stimulated ex vivo with 10 µg/ml CpG-A or CpG-B and 5 µg/ml R837. Bars show IFN-α, IL-6, and TNF protein levels determined by ELISA in cell culture supernatant. Results are from two (R837) to five (CpG-A and CpG-B) independent experiments each with six mice per group. (B) pDCs were stimulated with 20 µg/ml CpG-A for 5 h in the presence of brefeldin A. Intracellular staining was performed with antibodies against IFN-α and TNF. The plots are from one representative experiment of three performed. (C) Splenic pDCs from young WT or WKO mice were incubated with sera from WT ( n = 4) or WKO ( n = 4) adult mice. Data show the levels of IFN-α in cell culture supernatants subtracted of the values in control wells without cells. Data are representative of two experiments with six mice per group. (D and E) BM-pDCs or BM-cDCs were nucleofected with siRNA control (si-irr) or WASp-specific (si-WASp) RNA oligos. (D) 3 × 10 5 control or WASp-silenced BM-pDCs were stimulated with 15 µg/ml CpG-A or CpG-B. IFN-α and IL-6 protein levels were determined by ELISA. Results are from four experiments. (E) 3 × 10 5 control or WASp-silenced BM-cDCs were stimulated with 15 µg/ml CpG-B. IL-12p40 and IL-6 protein levels were determined by ELISA. Results are representative of three experiments. (F) 3 × 10 5 BM-pDCs were preincubated with 1 µg/ml cytochalasin- d (Cyto- d ), 0.1 µg/ml latrunculin-A or control medium, washed and stimulated with 5 µg/ml CpG-A for 5 h. Bars show concentrations of IFN-α and IL-6 protein in cell culture supernatants. Data are representative of two (latrunculin-A) to six (cytochalasin- d ) independent experiments. Levels of IL-6 (G) and IL-12p40 (H) induced by CpG-B in Cyto-D–treated pDCs and Cyto-D–treated cDCs upon stimulation with 15 µg/ml CpG-A and CpG-B, respectively. One representative experiment out of three with similar results is shown. A, D, and E, Student’s t test; F, Mann-Whitney test; error bars indicate SEM. *, P

Techniques Used: Mouse Assay, Isolation, Ex Vivo, Enzyme-linked Immunosorbent Assay, Cell Culture, Staining, Incubation, MANN-WHITNEY

20) Product Images from "Anti-hepatitis B virus (HBV) response of imiquimod based toll like receptor 7 ligand in hbv-positive human hepatocelluar carcinoma cell line"

Article Title: Anti-hepatitis B virus (HBV) response of imiquimod based toll like receptor 7 ligand in hbv-positive human hepatocelluar carcinoma cell line

Journal: BMC Infectious Diseases

doi: 10.1186/s12879-017-2189-z

Status of epigenetic markers and transcription factors on TLR7 activation. HepG2.2.15 cells were treated with R837 for 72 h and then the cells were subjected to Western Blot for protein expression studies. a Protein expression of different histone modifications (H3K4Me3, H3K9Me3, H3K36Me3 and H3K9Ac) and p53 before and after TLR7 activation. b The relative expression of the proteins (H3K9Me3 and p53) in HepG2 and HepG2.2.15 cells
Figure Legend Snippet: Status of epigenetic markers and transcription factors on TLR7 activation. HepG2.2.15 cells were treated with R837 for 72 h and then the cells were subjected to Western Blot for protein expression studies. a Protein expression of different histone modifications (H3K4Me3, H3K9Me3, H3K36Me3 and H3K9Ac) and p53 before and after TLR7 activation. b The relative expression of the proteins (H3K9Me3 and p53) in HepG2 and HepG2.2.15 cells

Techniques Used: Activation Assay, Western Blot, Expressing

Flow cytometric analysis and alteration of key cell cycle regulators upon TLR7 activation. HepG2.2.15 cells were treated with R837 (8μg/ml) for 72 h and then subjected to flow cytometric analysis to study the status of G1/S arrest. a Cell cycle analysis by FACS. b Percentage of cells in different phases of cell cycle before and after TLR7 activation. c p53 expression in liver biopsy samples of chronic HBV patients compared to control steatosis patients. d Relative mRNA expression of different cell cycle regulators (p53, Cyclin E, Cyclin D1, Cyclin B1 and Cyclin A) on R837 treatment. e Detection of invitro cell proliferation on TLR7 activation using MTT assay. HepG2.2.15 cells were treated with R837 for 72 h and then incubated with MTT at the indicated time points
Figure Legend Snippet: Flow cytometric analysis and alteration of key cell cycle regulators upon TLR7 activation. HepG2.2.15 cells were treated with R837 (8μg/ml) for 72 h and then subjected to flow cytometric analysis to study the status of G1/S arrest. a Cell cycle analysis by FACS. b Percentage of cells in different phases of cell cycle before and after TLR7 activation. c p53 expression in liver biopsy samples of chronic HBV patients compared to control steatosis patients. d Relative mRNA expression of different cell cycle regulators (p53, Cyclin E, Cyclin D1, Cyclin B1 and Cyclin A) on R837 treatment. e Detection of invitro cell proliferation on TLR7 activation using MTT assay. HepG2.2.15 cells were treated with R837 for 72 h and then incubated with MTT at the indicated time points

Techniques Used: Flow Cytometry, Activation Assay, Cell Cycle Assay, FACS, Expressing, MTT Assay, Incubation

TLR7 pathway is activated on stimulation with its specific ligand imiquimod, R837. Chief downstream regulators, especially NF-ĸB is activated and transclocated to the nucleus on TLR7 activation. a Host immune response on TLR7 activation, using TLR7 ligand. HepG2.2.15 cells were treated with TLR7 agonist, R837 (8μg/ml) for 3 days. mRNA expression of different downstream regulators (NF- КB, JNK, p38 and PI3K) involved in the TLR7 pathway. b Confocal imaging showing nuclear uptake of Nuclear Factor-КB p65 subunit (NF- КB) from cytoplasm in cells where HepG2.2.15 cells are taken as control and HepG2.2.15 cells stimulated with TLR7 ligand, R837 (8μg/ml) for 72 h. c Nuclear Factor-КB p65 subunit (NF-КB) protein expression of HepG2 and HepG2.2.15 cells. d Nuclear Factor-КB p65 subunit (NF- КB) protein expression of HepG2.2.15 cells taken as control and treated with R837 (8μg/ml) for 72 h
Figure Legend Snippet: TLR7 pathway is activated on stimulation with its specific ligand imiquimod, R837. Chief downstream regulators, especially NF-ĸB is activated and transclocated to the nucleus on TLR7 activation. a Host immune response on TLR7 activation, using TLR7 ligand. HepG2.2.15 cells were treated with TLR7 agonist, R837 (8μg/ml) for 3 days. mRNA expression of different downstream regulators (NF- КB, JNK, p38 and PI3K) involved in the TLR7 pathway. b Confocal imaging showing nuclear uptake of Nuclear Factor-КB p65 subunit (NF- КB) from cytoplasm in cells where HepG2.2.15 cells are taken as control and HepG2.2.15 cells stimulated with TLR7 ligand, R837 (8μg/ml) for 72 h. c Nuclear Factor-КB p65 subunit (NF-КB) protein expression of HepG2 and HepG2.2.15 cells. d Nuclear Factor-КB p65 subunit (NF- КB) protein expression of HepG2.2.15 cells taken as control and treated with R837 (8μg/ml) for 72 h

Techniques Used: Activation Assay, Expressing, Imaging

TLR7 mediated cellular signaling pathway in suppression of HBV replication. HepG2.2.15 cells were pretreated with or without different pathway inhibitors (NF-КB pathway inhibitor PDTC, MAPK/JNK pathway inhibitor SP600125, PI3K-AKT inhibitor LY294002 and MAPK/p38 pathway inhibitor SB203580). After 1 h incubation, R837 was treated for 6 h. a Schematic representation of TLR7 signaling pathway and important inhibitors of relevant signaling molecules. b and c . HBV DNA, HBsAg AND HBeAg from culture supernatants on stimulation with R837 alone or pre-treated with different pathway inhibitors detected by real time RT- PCR and ELISA. d . Relative mRNA expression of JNK–dependent/ independent responsive genes (c-Jun, c-Myc, Elk-1 and SAP-1)
Figure Legend Snippet: TLR7 mediated cellular signaling pathway in suppression of HBV replication. HepG2.2.15 cells were pretreated with or without different pathway inhibitors (NF-КB pathway inhibitor PDTC, MAPK/JNK pathway inhibitor SP600125, PI3K-AKT inhibitor LY294002 and MAPK/p38 pathway inhibitor SB203580). After 1 h incubation, R837 was treated for 6 h. a Schematic representation of TLR7 signaling pathway and important inhibitors of relevant signaling molecules. b and c . HBV DNA, HBsAg AND HBeAg from culture supernatants on stimulation with R837 alone or pre-treated with different pathway inhibitors detected by real time RT- PCR and ELISA. d . Relative mRNA expression of JNK–dependent/ independent responsive genes (c-Jun, c-Myc, Elk-1 and SAP-1)

Techniques Used: Incubation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing

HBV titre was evaluated in culture supernatants of HepG2.2.15 cells after treatment with 4, 6 and 8μg/ml of TLR7 agonist (R837) for 72 h. a HBV DNA was isolated from the culture media of R837 treated cells and the load was assessed by absolute real-time PCR using WHO standards. b HBsAg and HBeAg were detected from the culture supernatant of treated cells by ELISA
Figure Legend Snippet: HBV titre was evaluated in culture supernatants of HepG2.2.15 cells after treatment with 4, 6 and 8μg/ml of TLR7 agonist (R837) for 72 h. a HBV DNA was isolated from the culture media of R837 treated cells and the load was assessed by absolute real-time PCR using WHO standards. b HBsAg and HBeAg were detected from the culture supernatant of treated cells by ELISA

Techniques Used: Isolation, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

21) Product Images from "Anti-hepatitis B virus (HBV) response of imiquimod based toll like receptor 7 ligand in hbv-positive human hepatocelluar carcinoma cell line"

Article Title: Anti-hepatitis B virus (HBV) response of imiquimod based toll like receptor 7 ligand in hbv-positive human hepatocelluar carcinoma cell line

Journal: BMC Infectious Diseases

doi: 10.1186/s12879-017-2189-z

Status of epigenetic markers and transcription factors on TLR7 activation. HepG2.2.15 cells were treated with R837 for 72 h and then the cells were subjected to Western Blot for protein expression studies. a Protein expression of different histone modifications (H3K4Me3, H3K9Me3, H3K36Me3 and H3K9Ac) and p53 before and after TLR7 activation. b The relative expression of the proteins (H3K9Me3 and p53) in HepG2 and HepG2.2.15 cells
Figure Legend Snippet: Status of epigenetic markers and transcription factors on TLR7 activation. HepG2.2.15 cells were treated with R837 for 72 h and then the cells were subjected to Western Blot for protein expression studies. a Protein expression of different histone modifications (H3K4Me3, H3K9Me3, H3K36Me3 and H3K9Ac) and p53 before and after TLR7 activation. b The relative expression of the proteins (H3K9Me3 and p53) in HepG2 and HepG2.2.15 cells

Techniques Used: Activation Assay, Western Blot, Expressing

Flow cytometric analysis and alteration of key cell cycle regulators upon TLR7 activation. HepG2.2.15 cells were treated with R837 (8μg/ml) for 72 h and then subjected to flow cytometric analysis to study the status of G1/S arrest. a Cell cycle analysis by FACS. b Percentage of cells in different phases of cell cycle before and after TLR7 activation. c p53 expression in liver biopsy samples of chronic HBV patients compared to control steatosis patients. d Relative mRNA expression of different cell cycle regulators (p53, Cyclin E, Cyclin D1, Cyclin B1 and Cyclin A) on R837 treatment. e Detection of invitro cell proliferation on TLR7 activation using MTT assay. HepG2.2.15 cells were treated with R837 for 72 h and then incubated with MTT at the indicated time points
Figure Legend Snippet: Flow cytometric analysis and alteration of key cell cycle regulators upon TLR7 activation. HepG2.2.15 cells were treated with R837 (8μg/ml) for 72 h and then subjected to flow cytometric analysis to study the status of G1/S arrest. a Cell cycle analysis by FACS. b Percentage of cells in different phases of cell cycle before and after TLR7 activation. c p53 expression in liver biopsy samples of chronic HBV patients compared to control steatosis patients. d Relative mRNA expression of different cell cycle regulators (p53, Cyclin E, Cyclin D1, Cyclin B1 and Cyclin A) on R837 treatment. e Detection of invitro cell proliferation on TLR7 activation using MTT assay. HepG2.2.15 cells were treated with R837 for 72 h and then incubated with MTT at the indicated time points

Techniques Used: Flow Cytometry, Activation Assay, Cell Cycle Assay, FACS, Expressing, MTT Assay, Incubation

TLR7 pathway is activated on stimulation with its specific ligand imiquimod, R837. Chief downstream regulators, especially NF-ĸB is activated and transclocated to the nucleus on TLR7 activation. a Host immune response on TLR7 activation, using TLR7 ligand. HepG2.2.15 cells were treated with TLR7 agonist, R837 (8μg/ml) for 3 days. mRNA expression of different downstream regulators (NF- КB, JNK, p38 and PI3K) involved in the TLR7 pathway. b Confocal imaging showing nuclear uptake of Nuclear Factor-КB p65 subunit (NF- КB) from cytoplasm in cells where HepG2.2.15 cells are taken as control and HepG2.2.15 cells stimulated with TLR7 ligand, R837 (8μg/ml) for 72 h. c Nuclear Factor-КB p65 subunit (NF-КB) protein expression of HepG2 and HepG2.2.15 cells. d Nuclear Factor-КB p65 subunit (NF- КB) protein expression of HepG2.2.15 cells taken as control and treated with R837 (8μg/ml) for 72 h
Figure Legend Snippet: TLR7 pathway is activated on stimulation with its specific ligand imiquimod, R837. Chief downstream regulators, especially NF-ĸB is activated and transclocated to the nucleus on TLR7 activation. a Host immune response on TLR7 activation, using TLR7 ligand. HepG2.2.15 cells were treated with TLR7 agonist, R837 (8μg/ml) for 3 days. mRNA expression of different downstream regulators (NF- КB, JNK, p38 and PI3K) involved in the TLR7 pathway. b Confocal imaging showing nuclear uptake of Nuclear Factor-КB p65 subunit (NF- КB) from cytoplasm in cells where HepG2.2.15 cells are taken as control and HepG2.2.15 cells stimulated with TLR7 ligand, R837 (8μg/ml) for 72 h. c Nuclear Factor-КB p65 subunit (NF-КB) protein expression of HepG2 and HepG2.2.15 cells. d Nuclear Factor-КB p65 subunit (NF- КB) protein expression of HepG2.2.15 cells taken as control and treated with R837 (8μg/ml) for 72 h

Techniques Used: Activation Assay, Expressing, Imaging

TLR7 mediated cellular signaling pathway in suppression of HBV replication. HepG2.2.15 cells were pretreated with or without different pathway inhibitors (NF-КB pathway inhibitor PDTC, MAPK/JNK pathway inhibitor SP600125, PI3K-AKT inhibitor LY294002 and MAPK/p38 pathway inhibitor SB203580). After 1 h incubation, R837 was treated for 6 h. a Schematic representation of TLR7 signaling pathway and important inhibitors of relevant signaling molecules. b and c . HBV DNA, HBsAg AND HBeAg from culture supernatants on stimulation with R837 alone or pre-treated with different pathway inhibitors detected by real time RT- PCR and ELISA. d . Relative mRNA expression of JNK–dependent/ independent responsive genes (c-Jun, c-Myc, Elk-1 and SAP-1)
Figure Legend Snippet: TLR7 mediated cellular signaling pathway in suppression of HBV replication. HepG2.2.15 cells were pretreated with or without different pathway inhibitors (NF-КB pathway inhibitor PDTC, MAPK/JNK pathway inhibitor SP600125, PI3K-AKT inhibitor LY294002 and MAPK/p38 pathway inhibitor SB203580). After 1 h incubation, R837 was treated for 6 h. a Schematic representation of TLR7 signaling pathway and important inhibitors of relevant signaling molecules. b and c . HBV DNA, HBsAg AND HBeAg from culture supernatants on stimulation with R837 alone or pre-treated with different pathway inhibitors detected by real time RT- PCR and ELISA. d . Relative mRNA expression of JNK–dependent/ independent responsive genes (c-Jun, c-Myc, Elk-1 and SAP-1)

Techniques Used: Incubation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing

HBV titre was evaluated in culture supernatants of HepG2.2.15 cells after treatment with 4, 6 and 8μg/ml of TLR7 agonist (R837) for 72 h. a HBV DNA was isolated from the culture media of R837 treated cells and the load was assessed by absolute real-time PCR using WHO standards. b HBsAg and HBeAg were detected from the culture supernatant of treated cells by ELISA
Figure Legend Snippet: HBV titre was evaluated in culture supernatants of HepG2.2.15 cells after treatment with 4, 6 and 8μg/ml of TLR7 agonist (R837) for 72 h. a HBV DNA was isolated from the culture media of R837 treated cells and the load was assessed by absolute real-time PCR using WHO standards. b HBsAg and HBeAg were detected from the culture supernatant of treated cells by ELISA

Techniques Used: Isolation, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

22) Product Images from "HIV-1 Activates Macrophages Independent of Toll-Like Receptors"

Article Title: HIV-1 Activates Macrophages Independent of Toll-Like Receptors

Journal: PLoS ONE

doi: 10.1371/journal.pone.0003664

HIV-1 priming enhances macrophage cytokine production in response to TLR stimulation. Macrophages were treated with HIV-1 or media alone for 24 hours. Virus was allowed to spread in culture for an additional 9 days. Macrophages in the absence (gray bars) or presence (black bars) of spreading virus were treated with media, HIV-1, LPS, Poly(I:C), CL097, CL075, or R837 for 24 hours, and supernatant TNF (A), IL-6 (B), or IL-1β (C) levels were measured.
Figure Legend Snippet: HIV-1 priming enhances macrophage cytokine production in response to TLR stimulation. Macrophages were treated with HIV-1 or media alone for 24 hours. Virus was allowed to spread in culture for an additional 9 days. Macrophages in the absence (gray bars) or presence (black bars) of spreading virus were treated with media, HIV-1, LPS, Poly(I:C), CL097, CL075, or R837 for 24 hours, and supernatant TNF (A), IL-6 (B), or IL-1β (C) levels were measured.

Techniques Used:

23) Product Images from "Human Neutrophils Produce CCL23 in Response to Various TLR-Agonists and TNFα"

Article Title: Human Neutrophils Produce CCL23 in Response to Various TLR-Agonists and TNFα

Journal: Frontiers in Cellular and Infection Microbiology

doi: 10.3389/fcimb.2017.00176

Other agonists, besides R848, trigger the production of CCL23 by human neutrophils . Neutrophils (5 × 10 6 /ml) were cultured with or without 5–50 μM R837, 5 μM R848, 5 μM CL075, 0.1–10 μg/ml LPS, 1 μg/ml Pam3CSK4, 10 ng/ml TNFα, 10 nM fMLF, 10 ng/ml G-CSF, 10 ng/ml GM-CSF, 500 μg/ml β-Glucan, 500 μg/ml Curdlan, 100 ng/ml IL-18, and 100 ng/ml IL-33 for 24 h. Then, extracellular supernatants were collected to evaluate CCL23 (A) and CXCL8 (B) production by ELISA (mean ± SEM, n = 3–5). Asterisks stand for significant increases as compared to untreated cells: * P
Figure Legend Snippet: Other agonists, besides R848, trigger the production of CCL23 by human neutrophils . Neutrophils (5 × 10 6 /ml) were cultured with or without 5–50 μM R837, 5 μM R848, 5 μM CL075, 0.1–10 μg/ml LPS, 1 μg/ml Pam3CSK4, 10 ng/ml TNFα, 10 nM fMLF, 10 ng/ml G-CSF, 10 ng/ml GM-CSF, 500 μg/ml β-Glucan, 500 μg/ml Curdlan, 100 ng/ml IL-18, and 100 ng/ml IL-33 for 24 h. Then, extracellular supernatants were collected to evaluate CCL23 (A) and CXCL8 (B) production by ELISA (mean ± SEM, n = 3–5). Asterisks stand for significant increases as compared to untreated cells: * P

Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay

24) Product Images from "Circulating Plasma Extracellular Vesicles from Septic Mice Induce Inflammation via MicroRNA- and TLR7-Dependent Mechanisms"

Article Title: Circulating Plasma Extracellular Vesicles from Septic Mice Induce Inflammation via MicroRNA- and TLR7-Dependent Mechanisms

Journal: The Journal of Immunology Author Choice

doi: 10.4049/jimmunol.1801008

Plasma EVs from septic mice induce cytokine production through TLR7-MyD88 signaling. BMDM were isolated from WT and genetically modified mice (TLR7 −/− , TLR3 −/− , MyD88 −/− , Trif −/− ) and treated with sham EVs (20 μg/ml), CLP EVs (20 μg/ml), P3C (TLR2 ligand, 1 μg/ml), poly(I:C) (TLR3 ligands, 10 μg/ml), or R837 (TLR7 ligand, 0.25 μg/ml). Sixteen hours after the treatment, the culture media were collected and cytokines (MIP-2, IL-6) were measured by ELISA. ( A and B ) TLR7 but not TLR3 signaling contributed to CLP EV–induced cytokine production. ( C and D ) MyD88- but not Trif-deficiency abolished CLP EV–mediated MIP-2 and IL-6 production. Each bar represents triplicate samples with each experiment repeated twice. *** p
Figure Legend Snippet: Plasma EVs from septic mice induce cytokine production through TLR7-MyD88 signaling. BMDM were isolated from WT and genetically modified mice (TLR7 −/− , TLR3 −/− , MyD88 −/− , Trif −/− ) and treated with sham EVs (20 μg/ml), CLP EVs (20 μg/ml), P3C (TLR2 ligand, 1 μg/ml), poly(I:C) (TLR3 ligands, 10 μg/ml), or R837 (TLR7 ligand, 0.25 μg/ml). Sixteen hours after the treatment, the culture media were collected and cytokines (MIP-2, IL-6) were measured by ELISA. ( A and B ) TLR7 but not TLR3 signaling contributed to CLP EV–induced cytokine production. ( C and D ) MyD88- but not Trif-deficiency abolished CLP EV–mediated MIP-2 and IL-6 production. Each bar represents triplicate samples with each experiment repeated twice. *** p

Techniques Used: Mouse Assay, Isolation, Genetically Modified, Enzyme-linked Immunosorbent Assay

25) Product Images from "Manipulation of the Toll-Like Receptor 7 Signaling Pathway by Epstein-Barr Virus ▿"

Article Title: Manipulation of the Toll-Like Receptor 7 Signaling Pathway by Epstein-Barr Virus ▿

Journal:

doi: 10.1128/JVI.01122-07

EBV ameliorates negative growth effects following overstimulation of TLR7. Naive primary B cells were infected with EBV in medium without or with R837 (20 μg/ml) added initially at the time of infection (day 0) or 2 days postinfection (day 2)
Figure Legend Snippet: EBV ameliorates negative growth effects following overstimulation of TLR7. Naive primary B cells were infected with EBV in medium without or with R837 (20 μg/ml) added initially at the time of infection (day 0) or 2 days postinfection (day 2)

Techniques Used: Infection

EBV-mediated stimulation of TLR7 enhances B-cell proliferation. (A) Naive B cells were incubated with UV-inactivated virus in the presence or absence of R837 (2 μg/ml) for 3 days. (B) Naive B cells were infected with BXI virus in the presence
Figure Legend Snippet: EBV-mediated stimulation of TLR7 enhances B-cell proliferation. (A) Naive B cells were incubated with UV-inactivated virus in the presence or absence of R837 (2 μg/ml) for 3 days. (B) Naive B cells were infected with BXI virus in the presence

Techniques Used: Incubation, Infection

26) Product Images from "Inverse Immunological Responses Induced by Allergic Rhinitis and Head and Neck Squamous Cell Carcinoma"

Article Title: Inverse Immunological Responses Induced by Allergic Rhinitis and Head and Neck Squamous Cell Carcinoma

Journal: PLoS ONE

doi: 10.1371/journal.pone.0086796

Decreased peripheral blood mononuclear cells (PBMC) activation in patients with head and neck squamous cell carcinoma (HNSCC). PBMC were isolated from blood collected from healthy controls (n = 10), patients with an ongoing seasonal allergic rhinitis (AR; n = 11) and patients with HNSCC (n = 9), and cultured with or without Pam 3 CSK 4 (1 µg/ml), LPS (1 µg/ml), R837 (5 µg/ml) or CpG (0.3 µM) for 24 h. ( A ) Thereafter, the supernatants were analyzed for the secreted cytokine profile with Luminex Multiplex Immunoassay, 4 out of 17 investigated cytokines are shown, ( B ) and the cells were examined for the expression of CD25 and CD98 on CD4 positive T helper (Th) cells with flow cytometry. The results are presented as the non-stimulated value minus the TLR stimulated values. Grey colored samples were analyzed on a BD LSRFortessa, whereas the rest of the samples were investigated on a Beckman Coulter Navios flow cytometer. One representative histogram from the healthy controls, AR patients and HNSCC patients is displayed. The open histograms represent LPS stimulated cells, and the light grey histograms are denoting the non-stimulated samples. ( C ) RNA was extracted from peripheral PBMC, isolated from healthy individuals (n = 8), AR patients (n = 10), and HNSCC patients (n = 7), made into cDNA, and thereafter analyzed for COX-1 and COX-2 mRNA expression with real-time PCR. Data are given in relation to the housekeeping gene β-actin, 100 000×2 −ΔCt , and presented as mean ± SEM. MFI = mean fluorescence intensity; * p ≤0.05; ** p ≤0.01; *** p ≤0.001.
Figure Legend Snippet: Decreased peripheral blood mononuclear cells (PBMC) activation in patients with head and neck squamous cell carcinoma (HNSCC). PBMC were isolated from blood collected from healthy controls (n = 10), patients with an ongoing seasonal allergic rhinitis (AR; n = 11) and patients with HNSCC (n = 9), and cultured with or without Pam 3 CSK 4 (1 µg/ml), LPS (1 µg/ml), R837 (5 µg/ml) or CpG (0.3 µM) for 24 h. ( A ) Thereafter, the supernatants were analyzed for the secreted cytokine profile with Luminex Multiplex Immunoassay, 4 out of 17 investigated cytokines are shown, ( B ) and the cells were examined for the expression of CD25 and CD98 on CD4 positive T helper (Th) cells with flow cytometry. The results are presented as the non-stimulated value minus the TLR stimulated values. Grey colored samples were analyzed on a BD LSRFortessa, whereas the rest of the samples were investigated on a Beckman Coulter Navios flow cytometer. One representative histogram from the healthy controls, AR patients and HNSCC patients is displayed. The open histograms represent LPS stimulated cells, and the light grey histograms are denoting the non-stimulated samples. ( C ) RNA was extracted from peripheral PBMC, isolated from healthy individuals (n = 8), AR patients (n = 10), and HNSCC patients (n = 7), made into cDNA, and thereafter analyzed for COX-1 and COX-2 mRNA expression with real-time PCR. Data are given in relation to the housekeeping gene β-actin, 100 000×2 −ΔCt , and presented as mean ± SEM. MFI = mean fluorescence intensity; * p ≤0.05; ** p ≤0.01; *** p ≤0.001.

Techniques Used: Activation Assay, Isolation, Cell Culture, Luminex, Multiplex Assay, Expressing, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Fluorescence

Increased activation of polymorphonuclear leukocytes (PMN) in patients with head and neck squamous cell carcinoma (HNSCC). Blood was obtained from healthy controls (n = 10), patients with an ongoing seasonal allergic rhinitis (AR; n = 11) and patients with HNSCC (ELISA n = 9; FACS n = 10). PMN were isolated and cultured in the presence or absence of Pam 3 CSK 4 (1 µg/ml), LPS (1 µg/ml), R837 (5 µg/ml) or CpG (0.3 µM) for 4 and 24 h. ( A ) The cell free supernatants were then analyzed for IL-6 and IL-8 with ELISA, ( B ) and the CD16 positive cells were investigated for the expression of CD11b and CD69 using flow cytometry. The results are presented as the non-stimulated value minus the TLR stimulated values. Grey colored samples were analyzed on a BD LSRFortessa, whereas the rest of the samples were investigated on a Beckman Coulter Navios flow cytometer. ( C ) PMN from healthy individuals (n = 9) were incubated with or without culture medium supernatant from the HNSCC cell line FaDu, and stimulated with or without 1 µg/ml LPS for 4 h, and analyzed for IL-6 and IL-8 secretion with ELISA. The basal cytokine levels in the media were subtracted from the concentrations present in the PMN cultures. MFI = mean fluorescence intensity; * p ≤0.05; ** p ≤0.01; *** p ≤0.001.
Figure Legend Snippet: Increased activation of polymorphonuclear leukocytes (PMN) in patients with head and neck squamous cell carcinoma (HNSCC). Blood was obtained from healthy controls (n = 10), patients with an ongoing seasonal allergic rhinitis (AR; n = 11) and patients with HNSCC (ELISA n = 9; FACS n = 10). PMN were isolated and cultured in the presence or absence of Pam 3 CSK 4 (1 µg/ml), LPS (1 µg/ml), R837 (5 µg/ml) or CpG (0.3 µM) for 4 and 24 h. ( A ) The cell free supernatants were then analyzed for IL-6 and IL-8 with ELISA, ( B ) and the CD16 positive cells were investigated for the expression of CD11b and CD69 using flow cytometry. The results are presented as the non-stimulated value minus the TLR stimulated values. Grey colored samples were analyzed on a BD LSRFortessa, whereas the rest of the samples were investigated on a Beckman Coulter Navios flow cytometer. ( C ) PMN from healthy individuals (n = 9) were incubated with or without culture medium supernatant from the HNSCC cell line FaDu, and stimulated with or without 1 µg/ml LPS for 4 h, and analyzed for IL-6 and IL-8 secretion with ELISA. The basal cytokine levels in the media were subtracted from the concentrations present in the PMN cultures. MFI = mean fluorescence intensity; * p ≤0.05; ** p ≤0.01; *** p ≤0.001.

Techniques Used: Activation Assay, Enzyme-linked Immunosorbent Assay, FACS, Isolation, Cell Culture, Expressing, Flow Cytometry, Cytometry, Incubation, Fluorescence

27) Product Images from "Identification of a Novel Toll-like Receptor 7 Endogenous Ligand in Rheumatoid Arthritis Synovial Fluid That Can Provoke Arthritic Joint Inflammation"

Article Title: Identification of a Novel Toll-like Receptor 7 Endogenous Ligand in Rheumatoid Arthritis Synovial Fluid That Can Provoke Arthritic Joint Inflammation

Journal: Arthritis & rheumatology (Hoboken, N.J.)

doi: 10.1002/art.39544

Detection of accentuated microRNA let-7b (miR-let-7b) expression in rheumatoid arthritis (RA) synovial fluid (SF) macrophages. A, Copy numbers of miR-let-7b in normal (NL) plasma, RA plasma, osteoarthritis (OA) SF, and RA SF (n = 10–13 samples per group), as determined by real-time reverse transcription-polymerase chain reaction (RT-PCR). B and C, Exosomal secretion of miR-let-7b and miR-29a in peripheral blood (PB) T cells, in vitro-differentiated macrophages (Mac), and neutrophils (neutro) (n = 3–5 samples per group) ( B ) as well as in RA SF macrophages and RA synovial tissue (ST) fibroblasts (fibro) (n = 3 samples per group) ( C ). D and E, Copy numbers of miR-let-7b in RA SF macrophages ( D ) or RA ST ( E ). Samples (n = 3 per group) were left untreated (NT) or were treated for 6 hours with 1 µ M staurosporine to induce apoptosis or were subjected to repetitive freeze-thawing to promote necrosis. Exosomes released into the medium were extracted, and miR-let-7b copies were quantified. F, Levels of mRNA for tumor necrosis factor (TNF) and interleukin-6 (IL-6), as determined by real-time RT-PCR (n = 3 samples per group). RA PB in vitro-differentiated macrophages were left untreated (phosphate buffered saline [PBS] alone) or were treated for 6 hours with 5 µg/ml of miR-124, miR-147, or miR-let-7b. G and H, Western blot analysis of Toll-like receptor 7 (TLR-7) expression in control or knockdown cells and quantification of IL-6 ( G ) and IL-8 ( H ) transcription levels in in vitro-differentiated RA PB macrophages (n = 3 samples per group) after knockdown with small interfering RNA (siRNA). Cells were transfected for 72 hours with 100 n M control (Ctl-si) or TLR-7 (TLR7-si) siRNA and were left untreated or were stimulated for 6 hours with 1 µg/ml of R837 or with 5 µg/ml of miR-124, miR-147, or miR-let-7b. I, Western blot analysis and quantification of THP-1 cells transfected for 72 hours with 100 n M control, TLR-7, or TLR-8 siRNA Cells were left untreated or were stimulated for 6 hours with miR-let-7b, and IL-6 was quantified by real-time RT-PCR (n = 3 samples per group). The expression of TLRs 7 and 8 was markedly reduced after knockdown as compared to controls. Actin was used in Western blots as loading control. Fold change represents the increase over no treatment. Values are the mean ± SEM. * = P
Figure Legend Snippet: Detection of accentuated microRNA let-7b (miR-let-7b) expression in rheumatoid arthritis (RA) synovial fluid (SF) macrophages. A, Copy numbers of miR-let-7b in normal (NL) plasma, RA plasma, osteoarthritis (OA) SF, and RA SF (n = 10–13 samples per group), as determined by real-time reverse transcription-polymerase chain reaction (RT-PCR). B and C, Exosomal secretion of miR-let-7b and miR-29a in peripheral blood (PB) T cells, in vitro-differentiated macrophages (Mac), and neutrophils (neutro) (n = 3–5 samples per group) ( B ) as well as in RA SF macrophages and RA synovial tissue (ST) fibroblasts (fibro) (n = 3 samples per group) ( C ). D and E, Copy numbers of miR-let-7b in RA SF macrophages ( D ) or RA ST ( E ). Samples (n = 3 per group) were left untreated (NT) or were treated for 6 hours with 1 µ M staurosporine to induce apoptosis or were subjected to repetitive freeze-thawing to promote necrosis. Exosomes released into the medium were extracted, and miR-let-7b copies were quantified. F, Levels of mRNA for tumor necrosis factor (TNF) and interleukin-6 (IL-6), as determined by real-time RT-PCR (n = 3 samples per group). RA PB in vitro-differentiated macrophages were left untreated (phosphate buffered saline [PBS] alone) or were treated for 6 hours with 5 µg/ml of miR-124, miR-147, or miR-let-7b. G and H, Western blot analysis of Toll-like receptor 7 (TLR-7) expression in control or knockdown cells and quantification of IL-6 ( G ) and IL-8 ( H ) transcription levels in in vitro-differentiated RA PB macrophages (n = 3 samples per group) after knockdown with small interfering RNA (siRNA). Cells were transfected for 72 hours with 100 n M control (Ctl-si) or TLR-7 (TLR7-si) siRNA and were left untreated or were stimulated for 6 hours with 1 µg/ml of R837 or with 5 µg/ml of miR-124, miR-147, or miR-let-7b. I, Western blot analysis and quantification of THP-1 cells transfected for 72 hours with 100 n M control, TLR-7, or TLR-8 siRNA Cells were left untreated or were stimulated for 6 hours with miR-let-7b, and IL-6 was quantified by real-time RT-PCR (n = 3 samples per group). The expression of TLRs 7 and 8 was markedly reduced after knockdown as compared to controls. Actin was used in Western blots as loading control. Fold change represents the increase over no treatment. Values are the mean ± SEM. * = P

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, In Vitro, Quantitative RT-PCR, Western Blot, Small Interfering RNA, Transfection, CTL Assay

28) Product Images from "Identification of a Novel Toll-like Receptor 7 Endogenous Ligand in Rheumatoid Arthritis Synovial Fluid That Can Provoke Arthritic Joint Inflammation"

Article Title: Identification of a Novel Toll-like Receptor 7 Endogenous Ligand in Rheumatoid Arthritis Synovial Fluid That Can Provoke Arthritic Joint Inflammation

Journal: Arthritis & rheumatology (Hoboken, N.J.)

doi: 10.1002/art.39544

Detection of accentuated microRNA let-7b (miR-let-7b) expression in rheumatoid arthritis (RA) synovial fluid (SF) macrophages. A, Copy numbers of miR-let-7b in normal (NL) plasma, RA plasma, osteoarthritis (OA) SF, and RA SF (n = 10–13 samples per group), as determined by real-time reverse transcription-polymerase chain reaction (RT-PCR). B and C, Exosomal secretion of miR-let-7b and miR-29a in peripheral blood (PB) T cells, in vitro-differentiated macrophages (Mac), and neutrophils (neutro) (n = 3–5 samples per group) ( B ) as well as in RA SF macrophages and RA synovial tissue (ST) fibroblasts (fibro) (n = 3 samples per group) ( C ). D and E, Copy numbers of miR-let-7b in RA SF macrophages ( D ) or RA ST ( E ). Samples (n = 3 per group) were left untreated (NT) or were treated for 6 hours with 1 µ M staurosporine to induce apoptosis or were subjected to repetitive freeze-thawing to promote necrosis. Exosomes released into the medium were extracted, and miR-let-7b copies were quantified. F, Levels of mRNA for tumor necrosis factor (TNF) and interleukin-6 (IL-6), as determined by real-time RT-PCR (n = 3 samples per group). RA PB in vitro-differentiated macrophages were left untreated (phosphate buffered saline [PBS] alone) or were treated for 6 hours with 5 µg/ml of miR-124, miR-147, or miR-let-7b. G and H, Western blot analysis of Toll-like receptor 7 (TLR-7) expression in control or knockdown cells and quantification of IL-6 ( G ) and IL-8 ( H ) transcription levels in in vitro-differentiated RA PB macrophages (n = 3 samples per group) after knockdown with small interfering RNA (siRNA). Cells were transfected for 72 hours with 100 n M control (Ctl-si) or TLR-7 (TLR7-si) siRNA and were left untreated or were stimulated for 6 hours with 1 µg/ml of R837 or with 5 µg/ml of miR-124, miR-147, or miR-let-7b. I, Western blot analysis and quantification of THP-1 cells transfected for 72 hours with 100 n M control, TLR-7, or TLR-8 siRNA Cells were left untreated or were stimulated for 6 hours with miR-let-7b, and IL-6 was quantified by real-time RT-PCR (n = 3 samples per group). The expression of TLRs 7 and 8 was markedly reduced after knockdown as compared to controls. Actin was used in Western blots as loading control. Fold change represents the increase over no treatment. Values are the mean ± SEM. * = P
Figure Legend Snippet: Detection of accentuated microRNA let-7b (miR-let-7b) expression in rheumatoid arthritis (RA) synovial fluid (SF) macrophages. A, Copy numbers of miR-let-7b in normal (NL) plasma, RA plasma, osteoarthritis (OA) SF, and RA SF (n = 10–13 samples per group), as determined by real-time reverse transcription-polymerase chain reaction (RT-PCR). B and C, Exosomal secretion of miR-let-7b and miR-29a in peripheral blood (PB) T cells, in vitro-differentiated macrophages (Mac), and neutrophils (neutro) (n = 3–5 samples per group) ( B ) as well as in RA SF macrophages and RA synovial tissue (ST) fibroblasts (fibro) (n = 3 samples per group) ( C ). D and E, Copy numbers of miR-let-7b in RA SF macrophages ( D ) or RA ST ( E ). Samples (n = 3 per group) were left untreated (NT) or were treated for 6 hours with 1 µ M staurosporine to induce apoptosis or were subjected to repetitive freeze-thawing to promote necrosis. Exosomes released into the medium were extracted, and miR-let-7b copies were quantified. F, Levels of mRNA for tumor necrosis factor (TNF) and interleukin-6 (IL-6), as determined by real-time RT-PCR (n = 3 samples per group). RA PB in vitro-differentiated macrophages were left untreated (phosphate buffered saline [PBS] alone) or were treated for 6 hours with 5 µg/ml of miR-124, miR-147, or miR-let-7b. G and H, Western blot analysis of Toll-like receptor 7 (TLR-7) expression in control or knockdown cells and quantification of IL-6 ( G ) and IL-8 ( H ) transcription levels in in vitro-differentiated RA PB macrophages (n = 3 samples per group) after knockdown with small interfering RNA (siRNA). Cells were transfected for 72 hours with 100 n M control (Ctl-si) or TLR-7 (TLR7-si) siRNA and were left untreated or were stimulated for 6 hours with 1 µg/ml of R837 or with 5 µg/ml of miR-124, miR-147, or miR-let-7b. I, Western blot analysis and quantification of THP-1 cells transfected for 72 hours with 100 n M control, TLR-7, or TLR-8 siRNA Cells were left untreated or were stimulated for 6 hours with miR-let-7b, and IL-6 was quantified by real-time RT-PCR (n = 3 samples per group). The expression of TLRs 7 and 8 was markedly reduced after knockdown as compared to controls. Actin was used in Western blots as loading control. Fold change represents the increase over no treatment. Values are the mean ± SEM. * = P

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, In Vitro, Quantitative RT-PCR, Western Blot, Small Interfering RNA, Transfection, CTL Assay

29) Product Images from "The RNA-binding protein Mex3B is a coreceptor of Toll-like receptor 3 in innate antiviral response"

Article Title: The RNA-binding protein Mex3B is a coreceptor of Toll-like receptor 3 in innate antiviral response

Journal: Cell Research

doi: 10.1038/cr.2016.16

Mex3B is essential for TLR3-mediated signaling in MEFs, BMDMs and DCs. (A) Mex3B deficiency inhibits poly(I:C)-induced transcription of Ifnb1 gene in MEFs. MEFs (2 × 10 5 ) were left untreated, treated with poly(I:C) (20 μg/ml), TNFα (10 ng/ml) or IL-1β (10 ng/ml) for 3 h, or infected with SeV or VSV for 12 h. Total RNA was then extracted for qPCR analysis. (B) Effects of Mex3B deficiency on poly(I:C)-induced phosphorylation of IRF3 and IκBα in MEFs. MEFs (2 × 10 5 ) were left untreated, infected with SeV or treated with poly(I:C) for the indicated times. Cells were lysed and immunoblot was performed with the indicated antibodies. (C , D) Mex3B deficiency inhibits poly(I:C)-induced transcription of Ifnb1 gene in BMDMs (C) and DCs (D) . The cells (2 × 10 5 ) were left untreated, treated with poly(I:C) (20 μg/ml), R837 (20 μg/ml) or PGN (10 μg/ml) for 3 h or infected with SeV, VSV, EMCV or HSV for 12 h before qPCR analysis. Data are represented as mean ± SEM. * P
Figure Legend Snippet: Mex3B is essential for TLR3-mediated signaling in MEFs, BMDMs and DCs. (A) Mex3B deficiency inhibits poly(I:C)-induced transcription of Ifnb1 gene in MEFs. MEFs (2 × 10 5 ) were left untreated, treated with poly(I:C) (20 μg/ml), TNFα (10 ng/ml) or IL-1β (10 ng/ml) for 3 h, or infected with SeV or VSV for 12 h. Total RNA was then extracted for qPCR analysis. (B) Effects of Mex3B deficiency on poly(I:C)-induced phosphorylation of IRF3 and IκBα in MEFs. MEFs (2 × 10 5 ) were left untreated, infected with SeV or treated with poly(I:C) for the indicated times. Cells were lysed and immunoblot was performed with the indicated antibodies. (C , D) Mex3B deficiency inhibits poly(I:C)-induced transcription of Ifnb1 gene in BMDMs (C) and DCs (D) . The cells (2 × 10 5 ) were left untreated, treated with poly(I:C) (20 μg/ml), R837 (20 μg/ml) or PGN (10 μg/ml) for 3 h or infected with SeV, VSV, EMCV or HSV for 12 h before qPCR analysis. Data are represented as mean ± SEM. * P

Techniques Used: Infection, Real-time Polymerase Chain Reaction

30) Product Images from "Dose-dependent phorbol 12-myristate-13-acetate-mediated monocyte-to-macrophage differentiation induces unique proteomic signatures in THP-1 cells"

Article Title: Dose-dependent phorbol 12-myristate-13-acetate-mediated monocyte-to-macrophage differentiation induces unique proteomic signatures in THP-1 cells

Journal: bioRxiv

doi: 10.1101/2020.02.27.968016

Expression dynamics of genes involved in innate immune signaling in response to PMA (A) Heatmap showing the proteome fold change of the subset of genes involved in innate immune signaling. Scale indicates the level of expression (Log2-expression values, z-transformed, scaled). (B) Dynamic expression pattern to assess the expression levels of pro-inflammatory cytokine mRNA induced by TLR agonists: CL075-TLR7/8, CpG2006-TLR9, Flagellin-TLR5, FSL1- TLR2/6, LPS 0111:B4, and LPS K12 – TLR4, Pam3CSK4-TLR2/1, Poly I:C-TLR3, R837-TLR7, and R848-TLR7/8. Heatmap depicts Log10 fold change of normalized gene expression for pairwise comparisons of mRNA levels.
Figure Legend Snippet: Expression dynamics of genes involved in innate immune signaling in response to PMA (A) Heatmap showing the proteome fold change of the subset of genes involved in innate immune signaling. Scale indicates the level of expression (Log2-expression values, z-transformed, scaled). (B) Dynamic expression pattern to assess the expression levels of pro-inflammatory cytokine mRNA induced by TLR agonists: CL075-TLR7/8, CpG2006-TLR9, Flagellin-TLR5, FSL1- TLR2/6, LPS 0111:B4, and LPS K12 – TLR4, Pam3CSK4-TLR2/1, Poly I:C-TLR3, R837-TLR7, and R848-TLR7/8. Heatmap depicts Log10 fold change of normalized gene expression for pairwise comparisons of mRNA levels.

Techniques Used: Expressing, Transformation Assay

31) Product Images from "Anti-hepatitis B virus (HBV) response of imiquimod based toll like receptor 7 ligand in hbv-positive human hepatocelluar carcinoma cell line"

Article Title: Anti-hepatitis B virus (HBV) response of imiquimod based toll like receptor 7 ligand in hbv-positive human hepatocelluar carcinoma cell line

Journal: BMC Infectious Diseases

doi: 10.1186/s12879-017-2189-z

Status of epigenetic markers and transcription factors on TLR7 activation. HepG2.2.15 cells were treated with R837 for 72 h and then the cells were subjected to Western Blot for protein expression studies. a Protein expression of different histone modifications (H3K4Me3, H3K9Me3, H3K36Me3 and H3K9Ac) and p53 before and after TLR7 activation. b The relative expression of the proteins (H3K9Me3 and p53) in HepG2 and HepG2.2.15 cells
Figure Legend Snippet: Status of epigenetic markers and transcription factors on TLR7 activation. HepG2.2.15 cells were treated with R837 for 72 h and then the cells were subjected to Western Blot for protein expression studies. a Protein expression of different histone modifications (H3K4Me3, H3K9Me3, H3K36Me3 and H3K9Ac) and p53 before and after TLR7 activation. b The relative expression of the proteins (H3K9Me3 and p53) in HepG2 and HepG2.2.15 cells

Techniques Used: Activation Assay, Western Blot, Expressing

Flow cytometric analysis and alteration of key cell cycle regulators upon TLR7 activation. HepG2.2.15 cells were treated with R837 (8μg/ml) for 72 h and then subjected to flow cytometric analysis to study the status of G1/S arrest. a Cell cycle analysis by FACS. b Percentage of cells in different phases of cell cycle before and after TLR7 activation. c p53 expression in liver biopsy samples of chronic HBV patients compared to control steatosis patients. d Relative mRNA expression of different cell cycle regulators (p53, Cyclin E, Cyclin D1, Cyclin B1 and Cyclin A) on R837 treatment. e Detection of invitro cell proliferation on TLR7 activation using MTT assay. HepG2.2.15 cells were treated with R837 for 72 h and then incubated with MTT at the indicated time points
Figure Legend Snippet: Flow cytometric analysis and alteration of key cell cycle regulators upon TLR7 activation. HepG2.2.15 cells were treated with R837 (8μg/ml) for 72 h and then subjected to flow cytometric analysis to study the status of G1/S arrest. a Cell cycle analysis by FACS. b Percentage of cells in different phases of cell cycle before and after TLR7 activation. c p53 expression in liver biopsy samples of chronic HBV patients compared to control steatosis patients. d Relative mRNA expression of different cell cycle regulators (p53, Cyclin E, Cyclin D1, Cyclin B1 and Cyclin A) on R837 treatment. e Detection of invitro cell proliferation on TLR7 activation using MTT assay. HepG2.2.15 cells were treated with R837 for 72 h and then incubated with MTT at the indicated time points

Techniques Used: Flow Cytometry, Activation Assay, Cell Cycle Assay, FACS, Expressing, MTT Assay, Incubation

TLR7 pathway is activated on stimulation with its specific ligand imiquimod, R837. Chief downstream regulators, especially NF-ĸB is activated and transclocated to the nucleus on TLR7 activation. a Host immune response on TLR7 activation, using TLR7 ligand. HepG2.2.15 cells were treated with TLR7 agonist, R837 (8μg/ml) for 3 days. mRNA expression of different downstream regulators (NF- КB, JNK, p38 and PI3K) involved in the TLR7 pathway. b Confocal imaging showing nuclear uptake of Nuclear Factor-КB p65 subunit (NF- КB) from cytoplasm in cells where HepG2.2.15 cells are taken as control and HepG2.2.15 cells stimulated with TLR7 ligand, R837 (8μg/ml) for 72 h. c Nuclear Factor-КB p65 subunit (NF-КB) protein expression of HepG2 and HepG2.2.15 cells. d Nuclear Factor-КB p65 subunit (NF- КB) protein expression of HepG2.2.15 cells taken as control and treated with R837 (8μg/ml) for 72 h
Figure Legend Snippet: TLR7 pathway is activated on stimulation with its specific ligand imiquimod, R837. Chief downstream regulators, especially NF-ĸB is activated and transclocated to the nucleus on TLR7 activation. a Host immune response on TLR7 activation, using TLR7 ligand. HepG2.2.15 cells were treated with TLR7 agonist, R837 (8μg/ml) for 3 days. mRNA expression of different downstream regulators (NF- КB, JNK, p38 and PI3K) involved in the TLR7 pathway. b Confocal imaging showing nuclear uptake of Nuclear Factor-КB p65 subunit (NF- КB) from cytoplasm in cells where HepG2.2.15 cells are taken as control and HepG2.2.15 cells stimulated with TLR7 ligand, R837 (8μg/ml) for 72 h. c Nuclear Factor-КB p65 subunit (NF-КB) protein expression of HepG2 and HepG2.2.15 cells. d Nuclear Factor-КB p65 subunit (NF- КB) protein expression of HepG2.2.15 cells taken as control and treated with R837 (8μg/ml) for 72 h

Techniques Used: Activation Assay, Expressing, Imaging

TLR7 mediated cellular signaling pathway in suppression of HBV replication. HepG2.2.15 cells were pretreated with or without different pathway inhibitors (NF-КB pathway inhibitor PDTC, MAPK/JNK pathway inhibitor SP600125, PI3K-AKT inhibitor LY294002 and MAPK/p38 pathway inhibitor SB203580). After 1 h incubation, R837 was treated for 6 h. a Schematic representation of TLR7 signaling pathway and important inhibitors of relevant signaling molecules. b and c . HBV DNA, HBsAg AND HBeAg from culture supernatants on stimulation with R837 alone or pre-treated with different pathway inhibitors detected by real time RT- PCR and ELISA. d . Relative mRNA expression of JNK–dependent/ independent responsive genes (c-Jun, c-Myc, Elk-1 and SAP-1)
Figure Legend Snippet: TLR7 mediated cellular signaling pathway in suppression of HBV replication. HepG2.2.15 cells were pretreated with or without different pathway inhibitors (NF-КB pathway inhibitor PDTC, MAPK/JNK pathway inhibitor SP600125, PI3K-AKT inhibitor LY294002 and MAPK/p38 pathway inhibitor SB203580). After 1 h incubation, R837 was treated for 6 h. a Schematic representation of TLR7 signaling pathway and important inhibitors of relevant signaling molecules. b and c . HBV DNA, HBsAg AND HBeAg from culture supernatants on stimulation with R837 alone or pre-treated with different pathway inhibitors detected by real time RT- PCR and ELISA. d . Relative mRNA expression of JNK–dependent/ independent responsive genes (c-Jun, c-Myc, Elk-1 and SAP-1)

Techniques Used: Incubation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing

HBV titre was evaluated in culture supernatants of HepG2.2.15 cells after treatment with 4, 6 and 8μg/ml of TLR7 agonist (R837) for 72 h. a HBV DNA was isolated from the culture media of R837 treated cells and the load was assessed by absolute real-time PCR using WHO standards. b HBsAg and HBeAg were detected from the culture supernatant of treated cells by ELISA
Figure Legend Snippet: HBV titre was evaluated in culture supernatants of HepG2.2.15 cells after treatment with 4, 6 and 8μg/ml of TLR7 agonist (R837) for 72 h. a HBV DNA was isolated from the culture media of R837 treated cells and the load was assessed by absolute real-time PCR using WHO standards. b HBsAg and HBeAg were detected from the culture supernatant of treated cells by ELISA

Techniques Used: Isolation, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

32) Product Images from "IFNα enhances the production of IL-6 by human neutrophils activated via TLR8"

Article Title: IFNα enhances the production of IL-6 by human neutrophils activated via TLR8

Journal: Scientific Reports

doi: 10.1038/srep19674

TLR7 is not expressed in human neutrophils incubated with IFNα. ( a ) TLR7 (left panel) and TLR8 (right panel) mRNA expression either in ( a ), neutrophils cultured with or without 5 μM R848, 5 and 25 μM R837, in the presence or absence of 1000 U/ml IFNα for 20 h, or ( b ), pDCs cultured for 5 h with or without 5 μM R837 or 1000 U/ml IFNα. In ( a ), no significant effect by IFNα was found by 2-way ANOVA followed by Bonferroni’s post-test. ( c ) IL-6 and CXCL8 levels detectable in supernatants from neutrophils treated for 20 h with or without 1–50 μM R837, in the presence or absence of 1000 U/ml IFNα. ( d ) CXCL8 mRNA expression in neutrophils pretreated with 25 nM Bafilomycin A1 (BafA1) for 30 min and then incubated for 5 h with 100 ng/ml LPS, 5 μM R848 or 50 μM R837. Values in panels ( a,c,d ) stand for the mean ± SEM (n = 5), while panel ( b ) depicts a representative experiment out of three independent ones with similar results. Asterisks in panel ( c,d ) indicate a significant increase with respect to untreated cells, while # symbols in panel ( d ) indicate a significant inhibition exerted by BafA1: *p
Figure Legend Snippet: TLR7 is not expressed in human neutrophils incubated with IFNα. ( a ) TLR7 (left panel) and TLR8 (right panel) mRNA expression either in ( a ), neutrophils cultured with or without 5 μM R848, 5 and 25 μM R837, in the presence or absence of 1000 U/ml IFNα for 20 h, or ( b ), pDCs cultured for 5 h with or without 5 μM R837 or 1000 U/ml IFNα. In ( a ), no significant effect by IFNα was found by 2-way ANOVA followed by Bonferroni’s post-test. ( c ) IL-6 and CXCL8 levels detectable in supernatants from neutrophils treated for 20 h with or without 1–50 μM R837, in the presence or absence of 1000 U/ml IFNα. ( d ) CXCL8 mRNA expression in neutrophils pretreated with 25 nM Bafilomycin A1 (BafA1) for 30 min and then incubated for 5 h with 100 ng/ml LPS, 5 μM R848 or 50 μM R837. Values in panels ( a,c,d ) stand for the mean ± SEM (n = 5), while panel ( b ) depicts a representative experiment out of three independent ones with similar results. Asterisks in panel ( c,d ) indicate a significant increase with respect to untreated cells, while # symbols in panel ( d ) indicate a significant inhibition exerted by BafA1: *p

Techniques Used: Incubation, Expressing, Cell Culture, Inhibition

33) Product Images from "Oxidized mitochondrial nucleoids released by neutrophils drive type I interferon production in human lupus"

Article Title: Oxidized mitochondrial nucleoids released by neutrophils drive type I interferon production in human lupus

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20151876

Live neutrophils extrude mtDNA–protein complexes. (A) Neutrophil supernatants from three healthy donors (HD) were run on agarose gels. High molecular weight complexes (mtC) yield a single DNA band of ∼16 Kb upon digestion with proteinase K (PK). (B) Amplification of the mitochondrial gene ND1 , but not the nuclear gene GAPDH , from DNA isolated from live neutrophil supernatants. Total neutrophil DNA was used as control. (C) Neutrophil supernatant (Orig Sup) was immunoprecipitated with anti-dsDNA antibody. The beads (IP Beads) and the resulting supernatants (IP Sup) were analyzed by agarose gel (left) or by Western blot with anti-TFAM or anti-H3 antibodies (right). (D) Abundance of mitochondrial and genomic DNA was assessed on 5 ng of isolated DNA from live, NETotic, or necrotic neutrophil supernatants by Real-Time PCR (mtDNA Copy Number; top) or by conventional PCR (bottom). (E) LDH activity was measured in cell-free supernatants from live, NETotic, or necrotic neutrophil cultures. (F) Quantification of extruded mtDNA by Real-Time PCR in the presence of DPI (NADPH Inhibitor) or MT (mtROS scavenger). (G, left) Apoptosis progression in untreated or GM-CSF–treated neutrophils was assessed by TUNEL assay. The percentage of TUNEL + cells is shown. (right) The amount of extruded mtDNA was assessed by Real-Time PCR (mtDNA Copy Number). (H, left) Extruded mtDNA was quantified by Picogreen. (right) mtDNA Copy Number in total cell DNA was quantified by Real-Time PCR. (I) The presence of TFAM, MnSOD, and TOMM20 was assessed in concentrated cell-free supernatants from live or necrotic neutrophils by Western blot. Coomassie staining of the BSA band was used as loading control. * indicates nonspecific band. (J) Surface expression of TOMM20, LAMP1, or Rab7 was assessed by flow cytometry on permeabilized (Total) or nonpermeabilized (Surface) neutrophils. Open gray histograms represent the isotype control. Δ MFI (Mean Fluorescence Intensity) = MFI antibody – MFI isotype control. (K–L) Extruded mtDNA (K) and TOMM20 plasma membrane expression (L) were measured in neutrophils treated with R837 (TLR7 agonist) in the presence or absence of IRS661 (TLR7 inhibitor) or with ODN2216 (TLR9 agonist). Data in B, C, and I are representative of three independent experiments with n = 3. Bars represent mean ± SD from at least three independent experiments, with n = 3–8. *, P
Figure Legend Snippet: Live neutrophils extrude mtDNA–protein complexes. (A) Neutrophil supernatants from three healthy donors (HD) were run on agarose gels. High molecular weight complexes (mtC) yield a single DNA band of ∼16 Kb upon digestion with proteinase K (PK). (B) Amplification of the mitochondrial gene ND1 , but not the nuclear gene GAPDH , from DNA isolated from live neutrophil supernatants. Total neutrophil DNA was used as control. (C) Neutrophil supernatant (Orig Sup) was immunoprecipitated with anti-dsDNA antibody. The beads (IP Beads) and the resulting supernatants (IP Sup) were analyzed by agarose gel (left) or by Western blot with anti-TFAM or anti-H3 antibodies (right). (D) Abundance of mitochondrial and genomic DNA was assessed on 5 ng of isolated DNA from live, NETotic, or necrotic neutrophil supernatants by Real-Time PCR (mtDNA Copy Number; top) or by conventional PCR (bottom). (E) LDH activity was measured in cell-free supernatants from live, NETotic, or necrotic neutrophil cultures. (F) Quantification of extruded mtDNA by Real-Time PCR in the presence of DPI (NADPH Inhibitor) or MT (mtROS scavenger). (G, left) Apoptosis progression in untreated or GM-CSF–treated neutrophils was assessed by TUNEL assay. The percentage of TUNEL + cells is shown. (right) The amount of extruded mtDNA was assessed by Real-Time PCR (mtDNA Copy Number). (H, left) Extruded mtDNA was quantified by Picogreen. (right) mtDNA Copy Number in total cell DNA was quantified by Real-Time PCR. (I) The presence of TFAM, MnSOD, and TOMM20 was assessed in concentrated cell-free supernatants from live or necrotic neutrophils by Western blot. Coomassie staining of the BSA band was used as loading control. * indicates nonspecific band. (J) Surface expression of TOMM20, LAMP1, or Rab7 was assessed by flow cytometry on permeabilized (Total) or nonpermeabilized (Surface) neutrophils. Open gray histograms represent the isotype control. Δ MFI (Mean Fluorescence Intensity) = MFI antibody – MFI isotype control. (K–L) Extruded mtDNA (K) and TOMM20 plasma membrane expression (L) were measured in neutrophils treated with R837 (TLR7 agonist) in the presence or absence of IRS661 (TLR7 inhibitor) or with ODN2216 (TLR9 agonist). Data in B, C, and I are representative of three independent experiments with n = 3. Bars represent mean ± SD from at least three independent experiments, with n = 3–8. *, P

Techniques Used: Molecular Weight, Amplification, Isolation, Immunoprecipitation, Agarose Gel Electrophoresis, Western Blot, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Activity Assay, TUNEL Assay, Staining, Expressing, Flow Cytometry, Cytometry, Fluorescence

34) Product Images from "GM-CSF and IL-4 synergistically trigger dendritic cells to acquire retinoic acid-producing capacity"

Article Title: GM-CSF and IL-4 synergistically trigger dendritic cells to acquire retinoic acid-producing capacity

Journal: International Immunology

doi: 10.1093/intimm/dxp003

TLR-mediated maturation signals enhance Aldh1a2 expression and ALDHact in BM-DCs. Flt3L-induced BM-DCs (A–C) or SPL-DCs (D and E) were cultured with medium alone (none), sPGN (10 μg ml −1 ), poly(I:C) (1 μg ml −1 ), LPS (1 μg ml −1 ), R837 (1 μg ml −1 ) or CpG ODN 1826 (CpG; 1 μM) in the presence or absence of GM-CSF and/or IL-4 (10 ng ml −1 each). (A and D) Expression of Aldh1a2 was assessed by semi-quantitative real-time PCR. The mRNA expression of treated cells is expressed as the ‘fold induction’ relative to that of untreated cells (cultured with medium alone without GM-CSF or IL-4). (B and E) DCs were cultured with medium alone or LPS in the presence or absence of GM-CSF and IL-4, incubated with ALDEFLUOR and co-stained for CD11c and CD86 expression. (C) BM-DCs were cultured with LPS, GM-CSF and IL-4, incubated with ALDEFLUOR and co-stained for CD11c and B220, CD4, CD8α, CD11b, CD45RB, CD80, CD103, F4/80 or MHC class II expression. ALDHact + cells are shown as red dots. All data are representative of two independent experiments.
Figure Legend Snippet: TLR-mediated maturation signals enhance Aldh1a2 expression and ALDHact in BM-DCs. Flt3L-induced BM-DCs (A–C) or SPL-DCs (D and E) were cultured with medium alone (none), sPGN (10 μg ml −1 ), poly(I:C) (1 μg ml −1 ), LPS (1 μg ml −1 ), R837 (1 μg ml −1 ) or CpG ODN 1826 (CpG; 1 μM) in the presence or absence of GM-CSF and/or IL-4 (10 ng ml −1 each). (A and D) Expression of Aldh1a2 was assessed by semi-quantitative real-time PCR. The mRNA expression of treated cells is expressed as the ‘fold induction’ relative to that of untreated cells (cultured with medium alone without GM-CSF or IL-4). (B and E) DCs were cultured with medium alone or LPS in the presence or absence of GM-CSF and IL-4, incubated with ALDEFLUOR and co-stained for CD11c and CD86 expression. (C) BM-DCs were cultured with LPS, GM-CSF and IL-4, incubated with ALDEFLUOR and co-stained for CD11c and B220, CD4, CD8α, CD11b, CD45RB, CD80, CD103, F4/80 or MHC class II expression. ALDHact + cells are shown as red dots. All data are representative of two independent experiments.

Techniques Used: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Incubation, Staining

35) Product Images from "Identification of a Novel Toll-like Receptor 7 Endogenous Ligand in Rheumatoid Arthritis Synovial Fluid That Can Provoke Arthritic Joint Inflammation"

Article Title: Identification of a Novel Toll-like Receptor 7 Endogenous Ligand in Rheumatoid Arthritis Synovial Fluid That Can Provoke Arthritic Joint Inflammation

Journal: Arthritis & rheumatology (Hoboken, N.J.)

doi: 10.1002/art.39544

Detection of accentuated microRNA let-7b (miR-let-7b) expression in rheumatoid arthritis (RA) synovial fluid (SF) macrophages. A, Copy numbers of miR-let-7b in normal (NL) plasma, RA plasma, osteoarthritis (OA) SF, and RA SF (n = 10–13 samples per group), as determined by real-time reverse transcription-polymerase chain reaction (RT-PCR). B and C, Exosomal secretion of miR-let-7b and miR-29a in peripheral blood (PB) T cells, in vitro-differentiated macrophages (Mac), and neutrophils (neutro) (n = 3–5 samples per group) ( B ) as well as in RA SF macrophages and RA synovial tissue (ST) fibroblasts (fibro) (n = 3 samples per group) ( C ). D and E, Copy numbers of miR-let-7b in RA SF macrophages ( D ) or RA ST ( E ). Samples (n = 3 per group) were left untreated (NT) or were treated for 6 hours with 1 µ M staurosporine to induce apoptosis or were subjected to repetitive freeze-thawing to promote necrosis. Exosomes released into the medium were extracted, and miR-let-7b copies were quantified. F, Levels of mRNA for tumor necrosis factor (TNF) and interleukin-6 (IL-6), as determined by real-time RT-PCR (n = 3 samples per group). RA PB in vitro-differentiated macrophages were left untreated (phosphate buffered saline [PBS] alone) or were treated for 6 hours with 5 µg/ml of miR-124, miR-147, or miR-let-7b. G and H, Western blot analysis of Toll-like receptor 7 (TLR-7) expression in control or knockdown cells and quantification of IL-6 ( G ) and IL-8 ( H ) transcription levels in in vitro-differentiated RA PB macrophages (n = 3 samples per group) after knockdown with small interfering RNA (siRNA). Cells were transfected for 72 hours with 100 n M control (Ctl-si) or TLR-7 (TLR7-si) siRNA and were left untreated or were stimulated for 6 hours with 1 µg/ml of R837 or with 5 µg/ml of miR-124, miR-147, or miR-let-7b. I, Western blot analysis and quantification of THP-1 cells transfected for 72 hours with 100 n M control, TLR-7, or TLR-8 siRNA Cells were left untreated or were stimulated for 6 hours with miR-let-7b, and IL-6 was quantified by real-time RT-PCR (n = 3 samples per group). The expression of TLRs 7 and 8 was markedly reduced after knockdown as compared to controls. Actin was used in Western blots as loading control. Fold change represents the increase over no treatment. Values are the mean ± SEM. * = P
Figure Legend Snippet: Detection of accentuated microRNA let-7b (miR-let-7b) expression in rheumatoid arthritis (RA) synovial fluid (SF) macrophages. A, Copy numbers of miR-let-7b in normal (NL) plasma, RA plasma, osteoarthritis (OA) SF, and RA SF (n = 10–13 samples per group), as determined by real-time reverse transcription-polymerase chain reaction (RT-PCR). B and C, Exosomal secretion of miR-let-7b and miR-29a in peripheral blood (PB) T cells, in vitro-differentiated macrophages (Mac), and neutrophils (neutro) (n = 3–5 samples per group) ( B ) as well as in RA SF macrophages and RA synovial tissue (ST) fibroblasts (fibro) (n = 3 samples per group) ( C ). D and E, Copy numbers of miR-let-7b in RA SF macrophages ( D ) or RA ST ( E ). Samples (n = 3 per group) were left untreated (NT) or were treated for 6 hours with 1 µ M staurosporine to induce apoptosis or were subjected to repetitive freeze-thawing to promote necrosis. Exosomes released into the medium were extracted, and miR-let-7b copies were quantified. F, Levels of mRNA for tumor necrosis factor (TNF) and interleukin-6 (IL-6), as determined by real-time RT-PCR (n = 3 samples per group). RA PB in vitro-differentiated macrophages were left untreated (phosphate buffered saline [PBS] alone) or were treated for 6 hours with 5 µg/ml of miR-124, miR-147, or miR-let-7b. G and H, Western blot analysis of Toll-like receptor 7 (TLR-7) expression in control or knockdown cells and quantification of IL-6 ( G ) and IL-8 ( H ) transcription levels in in vitro-differentiated RA PB macrophages (n = 3 samples per group) after knockdown with small interfering RNA (siRNA). Cells were transfected for 72 hours with 100 n M control (Ctl-si) or TLR-7 (TLR7-si) siRNA and were left untreated or were stimulated for 6 hours with 1 µg/ml of R837 or with 5 µg/ml of miR-124, miR-147, or miR-let-7b. I, Western blot analysis and quantification of THP-1 cells transfected for 72 hours with 100 n M control, TLR-7, or TLR-8 siRNA Cells were left untreated or were stimulated for 6 hours with miR-let-7b, and IL-6 was quantified by real-time RT-PCR (n = 3 samples per group). The expression of TLRs 7 and 8 was markedly reduced after knockdown as compared to controls. Actin was used in Western blots as loading control. Fold change represents the increase over no treatment. Values are the mean ± SEM. * = P

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, In Vitro, Quantitative RT-PCR, Western Blot, Small Interfering RNA, Transfection, CTL Assay

36) Product Images from "Anti-hepatitis B virus (HBV) response of imiquimod based toll like receptor 7 ligand in hbv-positive human hepatocelluar carcinoma cell line"

Article Title: Anti-hepatitis B virus (HBV) response of imiquimod based toll like receptor 7 ligand in hbv-positive human hepatocelluar carcinoma cell line

Journal: BMC Infectious Diseases

doi: 10.1186/s12879-017-2189-z

Status of epigenetic markers and transcription factors on TLR7 activation. HepG2.2.15 cells were treated with R837 for 72 h and then the cells were subjected to Western Blot for protein expression studies. a Protein expression of different histone modifications (H3K4Me3, H3K9Me3, H3K36Me3 and H3K9Ac) and p53 before and after TLR7 activation. b The relative expression of the proteins (H3K9Me3 and p53) in HepG2 and HepG2.2.15 cells
Figure Legend Snippet: Status of epigenetic markers and transcription factors on TLR7 activation. HepG2.2.15 cells were treated with R837 for 72 h and then the cells were subjected to Western Blot for protein expression studies. a Protein expression of different histone modifications (H3K4Me3, H3K9Me3, H3K36Me3 and H3K9Ac) and p53 before and after TLR7 activation. b The relative expression of the proteins (H3K9Me3 and p53) in HepG2 and HepG2.2.15 cells

Techniques Used: Activation Assay, Western Blot, Expressing

Flow cytometric analysis and alteration of key cell cycle regulators upon TLR7 activation. HepG2.2.15 cells were treated with R837 (8μg/ml) for 72 h and then subjected to flow cytometric analysis to study the status of G1/S arrest. a Cell cycle analysis by FACS. b Percentage of cells in different phases of cell cycle before and after TLR7 activation. c p53 expression in liver biopsy samples of chronic HBV patients compared to control steatosis patients. d Relative mRNA expression of different cell cycle regulators (p53, Cyclin E, Cyclin D1, Cyclin B1 and Cyclin A) on R837 treatment. e Detection of invitro cell proliferation on TLR7 activation using MTT assay. HepG2.2.15 cells were treated with R837 for 72 h and then incubated with MTT at the indicated time points
Figure Legend Snippet: Flow cytometric analysis and alteration of key cell cycle regulators upon TLR7 activation. HepG2.2.15 cells were treated with R837 (8μg/ml) for 72 h and then subjected to flow cytometric analysis to study the status of G1/S arrest. a Cell cycle analysis by FACS. b Percentage of cells in different phases of cell cycle before and after TLR7 activation. c p53 expression in liver biopsy samples of chronic HBV patients compared to control steatosis patients. d Relative mRNA expression of different cell cycle regulators (p53, Cyclin E, Cyclin D1, Cyclin B1 and Cyclin A) on R837 treatment. e Detection of invitro cell proliferation on TLR7 activation using MTT assay. HepG2.2.15 cells were treated with R837 for 72 h and then incubated with MTT at the indicated time points

Techniques Used: Flow Cytometry, Activation Assay, Cell Cycle Assay, FACS, Expressing, MTT Assay, Incubation

TLR7 pathway is activated on stimulation with its specific ligand imiquimod, R837. Chief downstream regulators, especially NF-ĸB is activated and transclocated to the nucleus on TLR7 activation. a Host immune response on TLR7 activation, using TLR7 ligand. HepG2.2.15 cells were treated with TLR7 agonist, R837 (8μg/ml) for 3 days. mRNA expression of different downstream regulators (NF- КB, JNK, p38 and PI3K) involved in the TLR7 pathway. b Confocal imaging showing nuclear uptake of Nuclear Factor-КB p65 subunit (NF- КB) from cytoplasm in cells where HepG2.2.15 cells are taken as control and HepG2.2.15 cells stimulated with TLR7 ligand, R837 (8μg/ml) for 72 h. c Nuclear Factor-КB p65 subunit (NF-КB) protein expression of HepG2 and HepG2.2.15 cells. d Nuclear Factor-КB p65 subunit (NF- КB) protein expression of HepG2.2.15 cells taken as control and treated with R837 (8μg/ml) for 72 h
Figure Legend Snippet: TLR7 pathway is activated on stimulation with its specific ligand imiquimod, R837. Chief downstream regulators, especially NF-ĸB is activated and transclocated to the nucleus on TLR7 activation. a Host immune response on TLR7 activation, using TLR7 ligand. HepG2.2.15 cells were treated with TLR7 agonist, R837 (8μg/ml) for 3 days. mRNA expression of different downstream regulators (NF- КB, JNK, p38 and PI3K) involved in the TLR7 pathway. b Confocal imaging showing nuclear uptake of Nuclear Factor-КB p65 subunit (NF- КB) from cytoplasm in cells where HepG2.2.15 cells are taken as control and HepG2.2.15 cells stimulated with TLR7 ligand, R837 (8μg/ml) for 72 h. c Nuclear Factor-КB p65 subunit (NF-КB) protein expression of HepG2 and HepG2.2.15 cells. d Nuclear Factor-КB p65 subunit (NF- КB) protein expression of HepG2.2.15 cells taken as control and treated with R837 (8μg/ml) for 72 h

Techniques Used: Activation Assay, Expressing, Imaging

TLR7 mediated cellular signaling pathway in suppression of HBV replication. HepG2.2.15 cells were pretreated with or without different pathway inhibitors (NF-КB pathway inhibitor PDTC, MAPK/JNK pathway inhibitor SP600125, PI3K-AKT inhibitor LY294002 and MAPK/p38 pathway inhibitor SB203580). After 1 h incubation, R837 was treated for 6 h. a Schematic representation of TLR7 signaling pathway and important inhibitors of relevant signaling molecules. b and c . HBV DNA, HBsAg AND HBeAg from culture supernatants on stimulation with R837 alone or pre-treated with different pathway inhibitors detected by real time RT- PCR and ELISA. d . Relative mRNA expression of JNK–dependent/ independent responsive genes (c-Jun, c-Myc, Elk-1 and SAP-1)
Figure Legend Snippet: TLR7 mediated cellular signaling pathway in suppression of HBV replication. HepG2.2.15 cells were pretreated with or without different pathway inhibitors (NF-КB pathway inhibitor PDTC, MAPK/JNK pathway inhibitor SP600125, PI3K-AKT inhibitor LY294002 and MAPK/p38 pathway inhibitor SB203580). After 1 h incubation, R837 was treated for 6 h. a Schematic representation of TLR7 signaling pathway and important inhibitors of relevant signaling molecules. b and c . HBV DNA, HBsAg AND HBeAg from culture supernatants on stimulation with R837 alone or pre-treated with different pathway inhibitors detected by real time RT- PCR and ELISA. d . Relative mRNA expression of JNK–dependent/ independent responsive genes (c-Jun, c-Myc, Elk-1 and SAP-1)

Techniques Used: Incubation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing

HBV titre was evaluated in culture supernatants of HepG2.2.15 cells after treatment with 4, 6 and 8μg/ml of TLR7 agonist (R837) for 72 h. a HBV DNA was isolated from the culture media of R837 treated cells and the load was assessed by absolute real-time PCR using WHO standards. b HBsAg and HBeAg were detected from the culture supernatant of treated cells by ELISA
Figure Legend Snippet: HBV titre was evaluated in culture supernatants of HepG2.2.15 cells after treatment with 4, 6 and 8μg/ml of TLR7 agonist (R837) for 72 h. a HBV DNA was isolated from the culture media of R837 treated cells and the load was assessed by absolute real-time PCR using WHO standards. b HBsAg and HBeAg were detected from the culture supernatant of treated cells by ELISA

Techniques Used: Isolation, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

37) Product Images from "Anti-hepatitis B virus (HBV) response of imiquimod based toll like receptor 7 ligand in hbv-positive human hepatocelluar carcinoma cell line"

Article Title: Anti-hepatitis B virus (HBV) response of imiquimod based toll like receptor 7 ligand in hbv-positive human hepatocelluar carcinoma cell line

Journal: BMC Infectious Diseases

doi: 10.1186/s12879-017-2189-z

Status of epigenetic markers and transcription factors on TLR7 activation. HepG2.2.15 cells were treated with R837 for 72 h and then the cells were subjected to Western Blot for protein expression studies. a Protein expression of different histone modifications (H3K4Me3, H3K9Me3, H3K36Me3 and H3K9Ac) and p53 before and after TLR7 activation. b The relative expression of the proteins (H3K9Me3 and p53) in HepG2 and HepG2.2.15 cells
Figure Legend Snippet: Status of epigenetic markers and transcription factors on TLR7 activation. HepG2.2.15 cells were treated with R837 for 72 h and then the cells were subjected to Western Blot for protein expression studies. a Protein expression of different histone modifications (H3K4Me3, H3K9Me3, H3K36Me3 and H3K9Ac) and p53 before and after TLR7 activation. b The relative expression of the proteins (H3K9Me3 and p53) in HepG2 and HepG2.2.15 cells

Techniques Used: Activation Assay, Western Blot, Expressing

Flow cytometric analysis and alteration of key cell cycle regulators upon TLR7 activation. HepG2.2.15 cells were treated with R837 (8μg/ml) for 72 h and then subjected to flow cytometric analysis to study the status of G1/S arrest. a Cell cycle analysis by FACS. b Percentage of cells in different phases of cell cycle before and after TLR7 activation. c p53 expression in liver biopsy samples of chronic HBV patients compared to control steatosis patients. d Relative mRNA expression of different cell cycle regulators (p53, Cyclin E, Cyclin D1, Cyclin B1 and Cyclin A) on R837 treatment. e Detection of invitro cell proliferation on TLR7 activation using MTT assay. HepG2.2.15 cells were treated with R837 for 72 h and then incubated with MTT at the indicated time points
Figure Legend Snippet: Flow cytometric analysis and alteration of key cell cycle regulators upon TLR7 activation. HepG2.2.15 cells were treated with R837 (8μg/ml) for 72 h and then subjected to flow cytometric analysis to study the status of G1/S arrest. a Cell cycle analysis by FACS. b Percentage of cells in different phases of cell cycle before and after TLR7 activation. c p53 expression in liver biopsy samples of chronic HBV patients compared to control steatosis patients. d Relative mRNA expression of different cell cycle regulators (p53, Cyclin E, Cyclin D1, Cyclin B1 and Cyclin A) on R837 treatment. e Detection of invitro cell proliferation on TLR7 activation using MTT assay. HepG2.2.15 cells were treated with R837 for 72 h and then incubated with MTT at the indicated time points

Techniques Used: Flow Cytometry, Activation Assay, Cell Cycle Assay, FACS, Expressing, MTT Assay, Incubation

TLR7 pathway is activated on stimulation with its specific ligand imiquimod, R837. Chief downstream regulators, especially NF-ĸB is activated and transclocated to the nucleus on TLR7 activation. a Host immune response on TLR7 activation, using TLR7 ligand. HepG2.2.15 cells were treated with TLR7 agonist, R837 (8μg/ml) for 3 days. mRNA expression of different downstream regulators (NF- КB, JNK, p38 and PI3K) involved in the TLR7 pathway. b Confocal imaging showing nuclear uptake of Nuclear Factor-КB p65 subunit (NF- КB) from cytoplasm in cells where HepG2.2.15 cells are taken as control and HepG2.2.15 cells stimulated with TLR7 ligand, R837 (8μg/ml) for 72 h. c Nuclear Factor-КB p65 subunit (NF-КB) protein expression of HepG2 and HepG2.2.15 cells. d Nuclear Factor-КB p65 subunit (NF- КB) protein expression of HepG2.2.15 cells taken as control and treated with R837 (8μg/ml) for 72 h
Figure Legend Snippet: TLR7 pathway is activated on stimulation with its specific ligand imiquimod, R837. Chief downstream regulators, especially NF-ĸB is activated and transclocated to the nucleus on TLR7 activation. a Host immune response on TLR7 activation, using TLR7 ligand. HepG2.2.15 cells were treated with TLR7 agonist, R837 (8μg/ml) for 3 days. mRNA expression of different downstream regulators (NF- КB, JNK, p38 and PI3K) involved in the TLR7 pathway. b Confocal imaging showing nuclear uptake of Nuclear Factor-КB p65 subunit (NF- КB) from cytoplasm in cells where HepG2.2.15 cells are taken as control and HepG2.2.15 cells stimulated with TLR7 ligand, R837 (8μg/ml) for 72 h. c Nuclear Factor-КB p65 subunit (NF-КB) protein expression of HepG2 and HepG2.2.15 cells. d Nuclear Factor-КB p65 subunit (NF- КB) protein expression of HepG2.2.15 cells taken as control and treated with R837 (8μg/ml) for 72 h

Techniques Used: Activation Assay, Expressing, Imaging

TLR7 mediated cellular signaling pathway in suppression of HBV replication. HepG2.2.15 cells were pretreated with or without different pathway inhibitors (NF-КB pathway inhibitor PDTC, MAPK/JNK pathway inhibitor SP600125, PI3K-AKT inhibitor LY294002 and MAPK/p38 pathway inhibitor SB203580). After 1 h incubation, R837 was treated for 6 h. a Schematic representation of TLR7 signaling pathway and important inhibitors of relevant signaling molecules. b and c . HBV DNA, HBsAg AND HBeAg from culture supernatants on stimulation with R837 alone or pre-treated with different pathway inhibitors detected by real time RT- PCR and ELISA. d . Relative mRNA expression of JNK–dependent/ independent responsive genes (c-Jun, c-Myc, Elk-1 and SAP-1)
Figure Legend Snippet: TLR7 mediated cellular signaling pathway in suppression of HBV replication. HepG2.2.15 cells were pretreated with or without different pathway inhibitors (NF-КB pathway inhibitor PDTC, MAPK/JNK pathway inhibitor SP600125, PI3K-AKT inhibitor LY294002 and MAPK/p38 pathway inhibitor SB203580). After 1 h incubation, R837 was treated for 6 h. a Schematic representation of TLR7 signaling pathway and important inhibitors of relevant signaling molecules. b and c . HBV DNA, HBsAg AND HBeAg from culture supernatants on stimulation with R837 alone or pre-treated with different pathway inhibitors detected by real time RT- PCR and ELISA. d . Relative mRNA expression of JNK–dependent/ independent responsive genes (c-Jun, c-Myc, Elk-1 and SAP-1)

Techniques Used: Incubation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing

HBV titre was evaluated in culture supernatants of HepG2.2.15 cells after treatment with 4, 6 and 8μg/ml of TLR7 agonist (R837) for 72 h. a HBV DNA was isolated from the culture media of R837 treated cells and the load was assessed by absolute real-time PCR using WHO standards. b HBsAg and HBeAg were detected from the culture supernatant of treated cells by ELISA
Figure Legend Snippet: HBV titre was evaluated in culture supernatants of HepG2.2.15 cells after treatment with 4, 6 and 8μg/ml of TLR7 agonist (R837) for 72 h. a HBV DNA was isolated from the culture media of R837 treated cells and the load was assessed by absolute real-time PCR using WHO standards. b HBsAg and HBeAg were detected from the culture supernatant of treated cells by ELISA

Techniques Used: Isolation, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

38) Product Images from "TLR2 and TLR4 triggering exerts contrasting effects with regard to HIV-1 infection of human dendritic cells and subsequent virus transfer to CD4+ T cells"

Article Title: TLR2 and TLR4 triggering exerts contrasting effects with regard to HIV-1 infection of human dendritic cells and subsequent virus transfer to CD4+ T cells

Journal: Retrovirology

doi: 10.1186/1742-4690-6-42

TLR2 and 4 triggering modulates HIV-1 transfer between IM-MDDCs and CD4 + T cells . A) IM-MDDCs were either left untreated (mock) or stimulated for 2 hours with the following TLR agonists: Pam3Csk4 (5 μg/ml), LPS (0.1 μg/ml), flagellin (5 μg/ml), R837 (5 μg/ml) and unmethylated DNA (5 μg/ml). Cells were then pulsed with NL4-3/Bal env and co-cultured with autologous CD4 + T cells. Finally cell-free supernatants were harvested at 2, 3 and 6 days post-coculture (dpcc) and the viral content was assessed by a p24 assay. Data depicted in the left panel represent the mean ± standard deviations of quadruplicate samples from a representative single donor at 2 dpcc, whereas the kinetics of virus production for the same donor are displayed in the small insert. Results from multiple different donors are illustrated in the right panel (2 dpcc) (**: P
Figure Legend Snippet: TLR2 and 4 triggering modulates HIV-1 transfer between IM-MDDCs and CD4 + T cells . A) IM-MDDCs were either left untreated (mock) or stimulated for 2 hours with the following TLR agonists: Pam3Csk4 (5 μg/ml), LPS (0.1 μg/ml), flagellin (5 μg/ml), R837 (5 μg/ml) and unmethylated DNA (5 μg/ml). Cells were then pulsed with NL4-3/Bal env and co-cultured with autologous CD4 + T cells. Finally cell-free supernatants were harvested at 2, 3 and 6 days post-coculture (dpcc) and the viral content was assessed by a p24 assay. Data depicted in the left panel represent the mean ± standard deviations of quadruplicate samples from a representative single donor at 2 dpcc, whereas the kinetics of virus production for the same donor are displayed in the small insert. Results from multiple different donors are illustrated in the right panel (2 dpcc) (**: P

Techniques Used: Cell Culture

39) Product Images from "Preventative effects of the partial RANKL peptide MHP1-AcN in a mouse model of imiquimod-induced psoriasis"

Article Title: Preventative effects of the partial RANKL peptide MHP1-AcN in a mouse model of imiquimod-induced psoriasis

Journal: Scientific Reports

doi: 10.1038/s41598-019-51681-0

MHP1-AcN inhibited R837-induced IL-6 production in RAW 264.7 cells. RAW 264.7 cells were stimulated with R837 (1 µg/mL) in the presence or absence of MHP1-AcN (0.1, 1, or 10 µg/mL) at the same time for 24 h. Cultured medium was collected and analyzed for IL-6 production by ELISA. *P
Figure Legend Snippet: MHP1-AcN inhibited R837-induced IL-6 production in RAW 264.7 cells. RAW 264.7 cells were stimulated with R837 (1 µg/mL) in the presence or absence of MHP1-AcN (0.1, 1, or 10 µg/mL) at the same time for 24 h. Cultured medium was collected and analyzed for IL-6 production by ELISA. *P

Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay

40) Product Images from "Anti-hepatitis B virus (HBV) response of imiquimod based toll like receptor 7 ligand in hbv-positive human hepatocelluar carcinoma cell line"

Article Title: Anti-hepatitis B virus (HBV) response of imiquimod based toll like receptor 7 ligand in hbv-positive human hepatocelluar carcinoma cell line

Journal: BMC Infectious Diseases

doi: 10.1186/s12879-017-2189-z

Status of epigenetic markers and transcription factors on TLR7 activation. HepG2.2.15 cells were treated with R837 for 72 h and then the cells were subjected to Western Blot for protein expression studies. a Protein expression of different histone modifications (H3K4Me3, H3K9Me3, H3K36Me3 and H3K9Ac) and p53 before and after TLR7 activation. b The relative expression of the proteins (H3K9Me3 and p53) in HepG2 and HepG2.2.15 cells
Figure Legend Snippet: Status of epigenetic markers and transcription factors on TLR7 activation. HepG2.2.15 cells were treated with R837 for 72 h and then the cells were subjected to Western Blot for protein expression studies. a Protein expression of different histone modifications (H3K4Me3, H3K9Me3, H3K36Me3 and H3K9Ac) and p53 before and after TLR7 activation. b The relative expression of the proteins (H3K9Me3 and p53) in HepG2 and HepG2.2.15 cells

Techniques Used: Activation Assay, Western Blot, Expressing

Flow cytometric analysis and alteration of key cell cycle regulators upon TLR7 activation. HepG2.2.15 cells were treated with R837 (8μg/ml) for 72 h and then subjected to flow cytometric analysis to study the status of G1/S arrest. a Cell cycle analysis by FACS. b Percentage of cells in different phases of cell cycle before and after TLR7 activation. c p53 expression in liver biopsy samples of chronic HBV patients compared to control steatosis patients. d Relative mRNA expression of different cell cycle regulators (p53, Cyclin E, Cyclin D1, Cyclin B1 and Cyclin A) on R837 treatment. e Detection of invitro cell proliferation on TLR7 activation using MTT assay. HepG2.2.15 cells were treated with R837 for 72 h and then incubated with MTT at the indicated time points
Figure Legend Snippet: Flow cytometric analysis and alteration of key cell cycle regulators upon TLR7 activation. HepG2.2.15 cells were treated with R837 (8μg/ml) for 72 h and then subjected to flow cytometric analysis to study the status of G1/S arrest. a Cell cycle analysis by FACS. b Percentage of cells in different phases of cell cycle before and after TLR7 activation. c p53 expression in liver biopsy samples of chronic HBV patients compared to control steatosis patients. d Relative mRNA expression of different cell cycle regulators (p53, Cyclin E, Cyclin D1, Cyclin B1 and Cyclin A) on R837 treatment. e Detection of invitro cell proliferation on TLR7 activation using MTT assay. HepG2.2.15 cells were treated with R837 for 72 h and then incubated with MTT at the indicated time points

Techniques Used: Flow Cytometry, Activation Assay, Cell Cycle Assay, FACS, Expressing, MTT Assay, Incubation

TLR7 pathway is activated on stimulation with its specific ligand imiquimod, R837. Chief downstream regulators, especially NF-ĸB is activated and transclocated to the nucleus on TLR7 activation. a Host immune response on TLR7 activation, using TLR7 ligand. HepG2.2.15 cells were treated with TLR7 agonist, R837 (8μg/ml) for 3 days. mRNA expression of different downstream regulators (NF- КB, JNK, p38 and PI3K) involved in the TLR7 pathway. b Confocal imaging showing nuclear uptake of Nuclear Factor-КB p65 subunit (NF- КB) from cytoplasm in cells where HepG2.2.15 cells are taken as control and HepG2.2.15 cells stimulated with TLR7 ligand, R837 (8μg/ml) for 72 h. c Nuclear Factor-КB p65 subunit (NF-КB) protein expression of HepG2 and HepG2.2.15 cells. d Nuclear Factor-КB p65 subunit (NF- КB) protein expression of HepG2.2.15 cells taken as control and treated with R837 (8μg/ml) for 72 h
Figure Legend Snippet: TLR7 pathway is activated on stimulation with its specific ligand imiquimod, R837. Chief downstream regulators, especially NF-ĸB is activated and transclocated to the nucleus on TLR7 activation. a Host immune response on TLR7 activation, using TLR7 ligand. HepG2.2.15 cells were treated with TLR7 agonist, R837 (8μg/ml) for 3 days. mRNA expression of different downstream regulators (NF- КB, JNK, p38 and PI3K) involved in the TLR7 pathway. b Confocal imaging showing nuclear uptake of Nuclear Factor-КB p65 subunit (NF- КB) from cytoplasm in cells where HepG2.2.15 cells are taken as control and HepG2.2.15 cells stimulated with TLR7 ligand, R837 (8μg/ml) for 72 h. c Nuclear Factor-КB p65 subunit (NF-КB) protein expression of HepG2 and HepG2.2.15 cells. d Nuclear Factor-КB p65 subunit (NF- КB) protein expression of HepG2.2.15 cells taken as control and treated with R837 (8μg/ml) for 72 h

Techniques Used: Activation Assay, Expressing, Imaging

TLR7 mediated cellular signaling pathway in suppression of HBV replication. HepG2.2.15 cells were pretreated with or without different pathway inhibitors (NF-КB pathway inhibitor PDTC, MAPK/JNK pathway inhibitor SP600125, PI3K-AKT inhibitor LY294002 and MAPK/p38 pathway inhibitor SB203580). After 1 h incubation, R837 was treated for 6 h. a Schematic representation of TLR7 signaling pathway and important inhibitors of relevant signaling molecules. b and c . HBV DNA, HBsAg AND HBeAg from culture supernatants on stimulation with R837 alone or pre-treated with different pathway inhibitors detected by real time RT- PCR and ELISA. d . Relative mRNA expression of JNK–dependent/ independent responsive genes (c-Jun, c-Myc, Elk-1 and SAP-1)
Figure Legend Snippet: TLR7 mediated cellular signaling pathway in suppression of HBV replication. HepG2.2.15 cells were pretreated with or without different pathway inhibitors (NF-КB pathway inhibitor PDTC, MAPK/JNK pathway inhibitor SP600125, PI3K-AKT inhibitor LY294002 and MAPK/p38 pathway inhibitor SB203580). After 1 h incubation, R837 was treated for 6 h. a Schematic representation of TLR7 signaling pathway and important inhibitors of relevant signaling molecules. b and c . HBV DNA, HBsAg AND HBeAg from culture supernatants on stimulation with R837 alone or pre-treated with different pathway inhibitors detected by real time RT- PCR and ELISA. d . Relative mRNA expression of JNK–dependent/ independent responsive genes (c-Jun, c-Myc, Elk-1 and SAP-1)

Techniques Used: Incubation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing

HBV titre was evaluated in culture supernatants of HepG2.2.15 cells after treatment with 4, 6 and 8μg/ml of TLR7 agonist (R837) for 72 h. a HBV DNA was isolated from the culture media of R837 treated cells and the load was assessed by absolute real-time PCR using WHO standards. b HBsAg and HBeAg were detected from the culture supernatant of treated cells by ELISA
Figure Legend Snippet: HBV titre was evaluated in culture supernatants of HepG2.2.15 cells after treatment with 4, 6 and 8μg/ml of TLR7 agonist (R837) for 72 h. a HBV DNA was isolated from the culture media of R837 treated cells and the load was assessed by absolute real-time PCR using WHO standards. b HBsAg and HBeAg were detected from the culture supernatant of treated cells by ELISA

Techniques Used: Isolation, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Related Articles

Magnetic Beads:

Article Title: Innate Immune Response of Human Plasmacytoid Dendritic Cells to Poxvirus Infection Is Subverted by Vaccinia E3 via Its Z-DNA/RNA Binding Domain
Article Snippet: .. Reagents The commercial sources for reagents were as follows: CpG oligodeoxynucleotide ODN2216 (CpG2216) and imiquimod (Invivogen); chloroquine and PI3K inhibitor LY294002 (Sigma-Aldrich); Akt inhibitors VIII and X (Calbiochem); human IFN-α and murine IFN-α/β enzyme-linked immunosorbent assay (ELISA) kits (PBL Biomedical Laboratories); TNF ELISA kit (R & D Systems); anti-BDCA-4 conjugated magnetic beads, anti-BDCA-2 PE and anti-CD123 APC (Miltenyi Biotec); Flt3L, R & D systems; anti-CD11c-APC and anti-B220-APC-Cy7 antibodies, BD Pharmingen; anti-mPDCA-1-PE antibody, Miltenyi Biotec. .. Cell Preparation And Isolation Of Human Pdcs Healthy donors provided peripheral blood after signing informed consent for research specimen collection using protocols approved by the Institutional Review and Privacy Board of Memorial Hospital, Memorial Sloan-Kettering Cancer Center.

Isolation:

Article Title: Macrophage MMP10 Regulates TLR7-Mediated Tolerance
Article Snippet: .. For TLR7 activation, BMDM were stimulated with IMQ (InvivoGen, San Diego, CA) in a 1- or 2-hit model. For the 2-hit model, BMDM (4 × 105 in 12-well plate) were stimulated with 400 ng/ml IMQ in PBS for 4 h, washed, incubated in fresh medium for 18 h, then re-stimulated with 1 μg/ml IMQ for 4 (RNA isolation) or 16 h (protein analysis) before harvest of cells and media. ..

Enzyme-linked Immunosorbent Assay:

Article Title: Innate Immune Response of Human Plasmacytoid Dendritic Cells to Poxvirus Infection Is Subverted by Vaccinia E3 via Its Z-DNA/RNA Binding Domain
Article Snippet: .. Reagents The commercial sources for reagents were as follows: CpG oligodeoxynucleotide ODN2216 (CpG2216) and imiquimod (Invivogen); chloroquine and PI3K inhibitor LY294002 (Sigma-Aldrich); Akt inhibitors VIII and X (Calbiochem); human IFN-α and murine IFN-α/β enzyme-linked immunosorbent assay (ELISA) kits (PBL Biomedical Laboratories); TNF ELISA kit (R & D Systems); anti-BDCA-4 conjugated magnetic beads, anti-BDCA-2 PE and anti-CD123 APC (Miltenyi Biotec); Flt3L, R & D systems; anti-CD11c-APC and anti-B220-APC-Cy7 antibodies, BD Pharmingen; anti-mPDCA-1-PE antibody, Miltenyi Biotec. .. Cell Preparation And Isolation Of Human Pdcs Healthy donors provided peripheral blood after signing informed consent for research specimen collection using protocols approved by the Institutional Review and Privacy Board of Memorial Hospital, Memorial Sloan-Kettering Cancer Center.

Activation Assay:

Article Title: Macrophage MMP10 Regulates TLR7-Mediated Tolerance
Article Snippet: .. For TLR7 activation, BMDM were stimulated with IMQ (InvivoGen, San Diego, CA) in a 1- or 2-hit model. For the 2-hit model, BMDM (4 × 105 in 12-well plate) were stimulated with 400 ng/ml IMQ in PBS for 4 h, washed, incubated in fresh medium for 18 h, then re-stimulated with 1 μg/ml IMQ for 4 (RNA isolation) or 16 h (protein analysis) before harvest of cells and media. ..

Incubation:

Article Title: Macrophage MMP10 Regulates TLR7-Mediated Tolerance
Article Snippet: .. For TLR7 activation, BMDM were stimulated with IMQ (InvivoGen, San Diego, CA) in a 1- or 2-hit model. For the 2-hit model, BMDM (4 × 105 in 12-well plate) were stimulated with 400 ng/ml IMQ in PBS for 4 h, washed, incubated in fresh medium for 18 h, then re-stimulated with 1 μg/ml IMQ for 4 (RNA isolation) or 16 h (protein analysis) before harvest of cells and media. ..

other:

Article Title: Effect of Adjuvants on Responses to Skin Immunization by Microneedles Coated with Influenza Subunit Vaccine
Article Snippet: Polyinosinic-polycytidylic acid (poly(I:C)) and imiquimod were purchased from Invivogen (San Diego, CA).

Article Title: Unprocessed interleukin-36α regulates psoriasis-like skin inflammation in co-operation with interleukin-1
Article Snippet: Water-soluble imiquimod was from InvivoGen.

Plasmid Preparation:

Article Title: Immunomodulator-Based Enhancement of Anti Smallpox Immune Responses
Article Snippet: .. Animals were immunized three times, two weeks apart with a dose of 100 mg of DNA plasmid coding for the VVWR A27 protein, with and without 50 nmoles of imiquimod or resiquimod (Invivogen, San Diego, CA). .. Also, Naïve and backbone mice groups were included as controls.

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    InvivoGen r837
    M45 inhibits IκBα degradation, TNFα and IL-6 production during MCMV infection. (A) NIH-3T3 cells were infected with wt MCMV-GFP, an M45 deletion mutant (ΔM45) or a revertant virus (RM45) at an MOI of 10 and treated 5 h postinfection with the TLR2 agonist Pam 3 CSK 4 (Pam., 0.1 µg/ml), the TLR4 agonist LPS (10 µg/ml), or IL-1β (20 ng/ml) for the indicated times. Levels of the indicated proteins were analyzed by immunoblotting. (B) BMDMs were infected with wt MCMV-GFP, ΔM45, or RM45 at an MOI of 3 and stimulated 8 h postinfection for 16 h with the TLR7 agonist R848 (0.1 µM). TNFα and IL-6 levels in the supernatant were determined by ELISA (mean ± SD). (C) RAW264.7 macrophages were infected with wt MCMV-GFP or ΔM45 at an MOI of 0.1, stimulated 24 h postinfection for 4 hours with TLR agonists Pam 3 CSK 4 (Pam.) or Malp-2 (TLR2), LPS (TLR4), <t>R837</t> (TLR7), or CpG (TLR9), in the presence of brefeldin A. Cells were fixed, permeabilized, and stained with a TNFα-specific antibody. The percentages of TNFα-positive cells within infected (GFP-positive) cell populations were determined by FACS analysis (mean ± SD).
    R837, supplied by InvivoGen, used in various techniques. Bioz Stars score: 91/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/r837/product/InvivoGen
    Average 91 stars, based on 17 article reviews
    Price from $9.99 to $1999.99
    r837 - by Bioz Stars, 2020-08
    91/100 stars
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    99
    InvivoGen imiquimod r837
    TLR7 pathway is activated on stimulation with its specific ligand <t>imiquimod,</t> <t>R837.</t> Chief downstream regulators, especially NF-ĸB is activated and transclocated to the nucleus on TLR7 activation. a Host immune response on TLR7 activation, using TLR7 ligand. HepG2.2.15 cells were treated with TLR7 agonist, R837 (8μg/ml) for 3 days. mRNA expression of different downstream regulators (NF- КB, JNK, p38 and PI3K) involved in the TLR7 pathway. b Confocal imaging showing nuclear uptake of Nuclear Factor-КB p65 subunit (NF- КB) from cytoplasm in cells where HepG2.2.15 cells are taken as control and HepG2.2.15 cells stimulated with TLR7 ligand, R837 (8μg/ml) for 72 h. c Nuclear Factor-КB p65 subunit (NF-КB) protein expression of HepG2 and HepG2.2.15 cells. d Nuclear Factor-КB p65 subunit (NF- КB) protein expression of HepG2.2.15 cells taken as control and treated with R837 (8μg/ml) for 72 h
    Imiquimod R837, supplied by InvivoGen, used in various techniques. Bioz Stars score: 99/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/imiquimod r837/product/InvivoGen
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    imiquimod r837 - by Bioz Stars, 2020-08
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    M45 inhibits IκBα degradation, TNFα and IL-6 production during MCMV infection. (A) NIH-3T3 cells were infected with wt MCMV-GFP, an M45 deletion mutant (ΔM45) or a revertant virus (RM45) at an MOI of 10 and treated 5 h postinfection with the TLR2 agonist Pam 3 CSK 4 (Pam., 0.1 µg/ml), the TLR4 agonist LPS (10 µg/ml), or IL-1β (20 ng/ml) for the indicated times. Levels of the indicated proteins were analyzed by immunoblotting. (B) BMDMs were infected with wt MCMV-GFP, ΔM45, or RM45 at an MOI of 3 and stimulated 8 h postinfection for 16 h with the TLR7 agonist R848 (0.1 µM). TNFα and IL-6 levels in the supernatant were determined by ELISA (mean ± SD). (C) RAW264.7 macrophages were infected with wt MCMV-GFP or ΔM45 at an MOI of 0.1, stimulated 24 h postinfection for 4 hours with TLR agonists Pam 3 CSK 4 (Pam.) or Malp-2 (TLR2), LPS (TLR4), R837 (TLR7), or CpG (TLR9), in the presence of brefeldin A. Cells were fixed, permeabilized, and stained with a TNFα-specific antibody. The percentages of TNFα-positive cells within infected (GFP-positive) cell populations were determined by FACS analysis (mean ± SD).

    Journal: PLoS Pathogens

    Article Title: Viral Mediated Redirection of NEMO/IKK? to Autophagosomes Curtails the Inflammatory Cascade

    doi: 10.1371/journal.ppat.1002517

    Figure Lengend Snippet: M45 inhibits IκBα degradation, TNFα and IL-6 production during MCMV infection. (A) NIH-3T3 cells were infected with wt MCMV-GFP, an M45 deletion mutant (ΔM45) or a revertant virus (RM45) at an MOI of 10 and treated 5 h postinfection with the TLR2 agonist Pam 3 CSK 4 (Pam., 0.1 µg/ml), the TLR4 agonist LPS (10 µg/ml), or IL-1β (20 ng/ml) for the indicated times. Levels of the indicated proteins were analyzed by immunoblotting. (B) BMDMs were infected with wt MCMV-GFP, ΔM45, or RM45 at an MOI of 3 and stimulated 8 h postinfection for 16 h with the TLR7 agonist R848 (0.1 µM). TNFα and IL-6 levels in the supernatant were determined by ELISA (mean ± SD). (C) RAW264.7 macrophages were infected with wt MCMV-GFP or ΔM45 at an MOI of 0.1, stimulated 24 h postinfection for 4 hours with TLR agonists Pam 3 CSK 4 (Pam.) or Malp-2 (TLR2), LPS (TLR4), R837 (TLR7), or CpG (TLR9), in the presence of brefeldin A. Cells were fixed, permeabilized, and stained with a TNFα-specific antibody. The percentages of TNFα-positive cells within infected (GFP-positive) cell populations were determined by FACS analysis (mean ± SD).

    Article Snippet: The following receptor ligands were used: LPS, LTA-SA, CpG (ODN 1668 or ODN 1826), R837 (Invivogen), R848 (Enzo LifeSciences), IL-1β, Pam3 CSK4 (Imgenex), Malp-2 (Alexis Biochemicals), TNFα (Promokine).

    Techniques: Infection, Mutagenesis, Enzyme-linked Immunosorbent Assay, Staining, FACS

    Reduced uptake of soluble antigen by BMDC in the presence of TLR agonists. Granulocyte macrophage colony-stimulating factor (GM-CSF) BMDC were cultured for 4 h with OVA–Alexa555 (5 μg ml −1 ) in the presence of polyI:C (50 μg ml −1 ), CpG (0.5 μg ml −1 ), R837 (1 μg ml −1 ), LPS (1 μg ml −1 ) or left untreated. Cells were stained with anti-CD11c antibody and samples were acquired by flow cytometry. (A) The mean fluorescent intensity of cells for Alexa555 was determined after gating on the CD11c + cell population. The data of two independent representative experiments were pooled ( n = 4) and the standard deviation is indicated. (B) The frequency of OVA–Alexa555 + cells was determined after gating on the CD11c + cell population. The relative percentage of OVA–Alexa555 + BMDC in samples stimulated with TLR agonists is depicted in relation to the frequency of OVA–Alexa555 + BMDC in the absence of TLR stimulation (unst), which was set to 100%. The results of eight independent experiments were compiled and the standard deviation is indicated. (C) Mice were vaccinated intravenously with GM-CSF BMDC that had been pulsed overnight with OVA in the presence or absence of various TLR ligands. Seven days after vaccination, CFSE-labelled target cells were injected and antigen-specific lysis of peptide-pulsed targets cells versus unpulsed target cells was determined by flow cytometry the following day. The graph compiles data from two independent experiments ( n = 10). For statistical analysis, one-way analysis of variance in combination with Dunnett's (A and B) or Tukey's (C) multiple comparison test was used (** P

    Journal: International Immunology

    Article Title: PolyI:C-induced reduction in uptake of soluble antigen is independent of dendritic cell activation

    doi: 10.1093/intimm/dxp053

    Figure Lengend Snippet: Reduced uptake of soluble antigen by BMDC in the presence of TLR agonists. Granulocyte macrophage colony-stimulating factor (GM-CSF) BMDC were cultured for 4 h with OVA–Alexa555 (5 μg ml −1 ) in the presence of polyI:C (50 μg ml −1 ), CpG (0.5 μg ml −1 ), R837 (1 μg ml −1 ), LPS (1 μg ml −1 ) or left untreated. Cells were stained with anti-CD11c antibody and samples were acquired by flow cytometry. (A) The mean fluorescent intensity of cells for Alexa555 was determined after gating on the CD11c + cell population. The data of two independent representative experiments were pooled ( n = 4) and the standard deviation is indicated. (B) The frequency of OVA–Alexa555 + cells was determined after gating on the CD11c + cell population. The relative percentage of OVA–Alexa555 + BMDC in samples stimulated with TLR agonists is depicted in relation to the frequency of OVA–Alexa555 + BMDC in the absence of TLR stimulation (unst), which was set to 100%. The results of eight independent experiments were compiled and the standard deviation is indicated. (C) Mice were vaccinated intravenously with GM-CSF BMDC that had been pulsed overnight with OVA in the presence or absence of various TLR ligands. Seven days after vaccination, CFSE-labelled target cells were injected and antigen-specific lysis of peptide-pulsed targets cells versus unpulsed target cells was determined by flow cytometry the following day. The graph compiles data from two independent experiments ( n = 10). For statistical analysis, one-way analysis of variance in combination with Dunnett's (A and B) or Tukey's (C) multiple comparison test was used (** P

    Article Snippet: PolyI:C was from GE Healthcare (Chalfont St Giles, UK), R837 and LPS (from Escherichia coli K12) were from Invivogen (Toulouse, France) and CpG-containing oligonucleotide (ODN) 1668 was purchased from MWG (Edersberg, Germany).

    Techniques: Cell Culture, Staining, Flow Cytometry, Cytometry, Standard Deviation, Mouse Assay, Injection, Lysis

    Status of epigenetic markers and transcription factors on TLR7 activation. HepG2.2.15 cells were treated with R837 for 72 h and then the cells were subjected to Western Blot for protein expression studies. a Protein expression of different histone modifications (H3K4Me3, H3K9Me3, H3K36Me3 and H3K9Ac) and p53 before and after TLR7 activation. b The relative expression of the proteins (H3K9Me3 and p53) in HepG2 and HepG2.2.15 cells

    Journal: BMC Infectious Diseases

    Article Title: Anti-hepatitis B virus (HBV) response of imiquimod based toll like receptor 7 ligand in hbv-positive human hepatocelluar carcinoma cell line

    doi: 10.1186/s12879-017-2189-z

    Figure Lengend Snippet: Status of epigenetic markers and transcription factors on TLR7 activation. HepG2.2.15 cells were treated with R837 for 72 h and then the cells were subjected to Western Blot for protein expression studies. a Protein expression of different histone modifications (H3K4Me3, H3K9Me3, H3K36Me3 and H3K9Ac) and p53 before and after TLR7 activation. b The relative expression of the proteins (H3K9Me3 and p53) in HepG2 and HepG2.2.15 cells

    Article Snippet: After 24 h, the media was removed and replaced with fresh media and treated with R837.

    Techniques: Activation Assay, Western Blot, Expressing

    Flow cytometric analysis and alteration of key cell cycle regulators upon TLR7 activation. HepG2.2.15 cells were treated with R837 (8μg/ml) for 72 h and then subjected to flow cytometric analysis to study the status of G1/S arrest. a Cell cycle analysis by FACS. b Percentage of cells in different phases of cell cycle before and after TLR7 activation. c p53 expression in liver biopsy samples of chronic HBV patients compared to control steatosis patients. d Relative mRNA expression of different cell cycle regulators (p53, Cyclin E, Cyclin D1, Cyclin B1 and Cyclin A) on R837 treatment. e Detection of invitro cell proliferation on TLR7 activation using MTT assay. HepG2.2.15 cells were treated with R837 for 72 h and then incubated with MTT at the indicated time points

    Journal: BMC Infectious Diseases

    Article Title: Anti-hepatitis B virus (HBV) response of imiquimod based toll like receptor 7 ligand in hbv-positive human hepatocelluar carcinoma cell line

    doi: 10.1186/s12879-017-2189-z

    Figure Lengend Snippet: Flow cytometric analysis and alteration of key cell cycle regulators upon TLR7 activation. HepG2.2.15 cells were treated with R837 (8μg/ml) for 72 h and then subjected to flow cytometric analysis to study the status of G1/S arrest. a Cell cycle analysis by FACS. b Percentage of cells in different phases of cell cycle before and after TLR7 activation. c p53 expression in liver biopsy samples of chronic HBV patients compared to control steatosis patients. d Relative mRNA expression of different cell cycle regulators (p53, Cyclin E, Cyclin D1, Cyclin B1 and Cyclin A) on R837 treatment. e Detection of invitro cell proliferation on TLR7 activation using MTT assay. HepG2.2.15 cells were treated with R837 for 72 h and then incubated with MTT at the indicated time points

    Article Snippet: After 24 h, the media was removed and replaced with fresh media and treated with R837.

    Techniques: Flow Cytometry, Activation Assay, Cell Cycle Assay, FACS, Expressing, MTT Assay, Incubation

    TLR7 pathway is activated on stimulation with its specific ligand imiquimod, R837. Chief downstream regulators, especially NF-ĸB is activated and transclocated to the nucleus on TLR7 activation. a Host immune response on TLR7 activation, using TLR7 ligand. HepG2.2.15 cells were treated with TLR7 agonist, R837 (8μg/ml) for 3 days. mRNA expression of different downstream regulators (NF- КB, JNK, p38 and PI3K) involved in the TLR7 pathway. b Confocal imaging showing nuclear uptake of Nuclear Factor-КB p65 subunit (NF- КB) from cytoplasm in cells where HepG2.2.15 cells are taken as control and HepG2.2.15 cells stimulated with TLR7 ligand, R837 (8μg/ml) for 72 h. c Nuclear Factor-КB p65 subunit (NF-КB) protein expression of HepG2 and HepG2.2.15 cells. d Nuclear Factor-КB p65 subunit (NF- КB) protein expression of HepG2.2.15 cells taken as control and treated with R837 (8μg/ml) for 72 h

    Journal: BMC Infectious Diseases

    Article Title: Anti-hepatitis B virus (HBV) response of imiquimod based toll like receptor 7 ligand in hbv-positive human hepatocelluar carcinoma cell line

    doi: 10.1186/s12879-017-2189-z

    Figure Lengend Snippet: TLR7 pathway is activated on stimulation with its specific ligand imiquimod, R837. Chief downstream regulators, especially NF-ĸB is activated and transclocated to the nucleus on TLR7 activation. a Host immune response on TLR7 activation, using TLR7 ligand. HepG2.2.15 cells were treated with TLR7 agonist, R837 (8μg/ml) for 3 days. mRNA expression of different downstream regulators (NF- КB, JNK, p38 and PI3K) involved in the TLR7 pathway. b Confocal imaging showing nuclear uptake of Nuclear Factor-КB p65 subunit (NF- КB) from cytoplasm in cells where HepG2.2.15 cells are taken as control and HepG2.2.15 cells stimulated with TLR7 ligand, R837 (8μg/ml) for 72 h. c Nuclear Factor-КB p65 subunit (NF-КB) protein expression of HepG2 and HepG2.2.15 cells. d Nuclear Factor-КB p65 subunit (NF- КB) protein expression of HepG2.2.15 cells taken as control and treated with R837 (8μg/ml) for 72 h

    Article Snippet: After 24 h, the media was removed and replaced with fresh media and treated with R837.

    Techniques: Activation Assay, Expressing, Imaging

    TLR7 mediated cellular signaling pathway in suppression of HBV replication. HepG2.2.15 cells were pretreated with or without different pathway inhibitors (NF-КB pathway inhibitor PDTC, MAPK/JNK pathway inhibitor SP600125, PI3K-AKT inhibitor LY294002 and MAPK/p38 pathway inhibitor SB203580). After 1 h incubation, R837 was treated for 6 h. a Schematic representation of TLR7 signaling pathway and important inhibitors of relevant signaling molecules. b and c . HBV DNA, HBsAg AND HBeAg from culture supernatants on stimulation with R837 alone or pre-treated with different pathway inhibitors detected by real time RT- PCR and ELISA. d . Relative mRNA expression of JNK–dependent/ independent responsive genes (c-Jun, c-Myc, Elk-1 and SAP-1)

    Journal: BMC Infectious Diseases

    Article Title: Anti-hepatitis B virus (HBV) response of imiquimod based toll like receptor 7 ligand in hbv-positive human hepatocelluar carcinoma cell line

    doi: 10.1186/s12879-017-2189-z

    Figure Lengend Snippet: TLR7 mediated cellular signaling pathway in suppression of HBV replication. HepG2.2.15 cells were pretreated with or without different pathway inhibitors (NF-КB pathway inhibitor PDTC, MAPK/JNK pathway inhibitor SP600125, PI3K-AKT inhibitor LY294002 and MAPK/p38 pathway inhibitor SB203580). After 1 h incubation, R837 was treated for 6 h. a Schematic representation of TLR7 signaling pathway and important inhibitors of relevant signaling molecules. b and c . HBV DNA, HBsAg AND HBeAg from culture supernatants on stimulation with R837 alone or pre-treated with different pathway inhibitors detected by real time RT- PCR and ELISA. d . Relative mRNA expression of JNK–dependent/ independent responsive genes (c-Jun, c-Myc, Elk-1 and SAP-1)

    Article Snippet: After 24 h, the media was removed and replaced with fresh media and treated with R837.

    Techniques: Incubation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing

    HBV titre was evaluated in culture supernatants of HepG2.2.15 cells after treatment with 4, 6 and 8μg/ml of TLR7 agonist (R837) for 72 h. a HBV DNA was isolated from the culture media of R837 treated cells and the load was assessed by absolute real-time PCR using WHO standards. b HBsAg and HBeAg were detected from the culture supernatant of treated cells by ELISA

    Journal: BMC Infectious Diseases

    Article Title: Anti-hepatitis B virus (HBV) response of imiquimod based toll like receptor 7 ligand in hbv-positive human hepatocelluar carcinoma cell line

    doi: 10.1186/s12879-017-2189-z

    Figure Lengend Snippet: HBV titre was evaluated in culture supernatants of HepG2.2.15 cells after treatment with 4, 6 and 8μg/ml of TLR7 agonist (R837) for 72 h. a HBV DNA was isolated from the culture media of R837 treated cells and the load was assessed by absolute real-time PCR using WHO standards. b HBsAg and HBeAg were detected from the culture supernatant of treated cells by ELISA

    Article Snippet: After 24 h, the media was removed and replaced with fresh media and treated with R837.

    Techniques: Isolation, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    TLR7 pathway is activated on stimulation with its specific ligand imiquimod, R837. Chief downstream regulators, especially NF-ĸB is activated and transclocated to the nucleus on TLR7 activation. a Host immune response on TLR7 activation, using TLR7 ligand. HepG2.2.15 cells were treated with TLR7 agonist, R837 (8μg/ml) for 3 days. mRNA expression of different downstream regulators (NF- КB, JNK, p38 and PI3K) involved in the TLR7 pathway. b Confocal imaging showing nuclear uptake of Nuclear Factor-КB p65 subunit (NF- КB) from cytoplasm in cells where HepG2.2.15 cells are taken as control and HepG2.2.15 cells stimulated with TLR7 ligand, R837 (8μg/ml) for 72 h. c Nuclear Factor-КB p65 subunit (NF-КB) protein expression of HepG2 and HepG2.2.15 cells. d Nuclear Factor-КB p65 subunit (NF- КB) protein expression of HepG2.2.15 cells taken as control and treated with R837 (8μg/ml) for 72 h

    Journal: BMC Infectious Diseases

    Article Title: Anti-hepatitis B virus (HBV) response of imiquimod based toll like receptor 7 ligand in hbv-positive human hepatocelluar carcinoma cell line

    doi: 10.1186/s12879-017-2189-z

    Figure Lengend Snippet: TLR7 pathway is activated on stimulation with its specific ligand imiquimod, R837. Chief downstream regulators, especially NF-ĸB is activated and transclocated to the nucleus on TLR7 activation. a Host immune response on TLR7 activation, using TLR7 ligand. HepG2.2.15 cells were treated with TLR7 agonist, R837 (8μg/ml) for 3 days. mRNA expression of different downstream regulators (NF- КB, JNK, p38 and PI3K) involved in the TLR7 pathway. b Confocal imaging showing nuclear uptake of Nuclear Factor-КB p65 subunit (NF- КB) from cytoplasm in cells where HepG2.2.15 cells are taken as control and HepG2.2.15 cells stimulated with TLR7 ligand, R837 (8μg/ml) for 72 h. c Nuclear Factor-КB p65 subunit (NF-КB) protein expression of HepG2 and HepG2.2.15 cells. d Nuclear Factor-КB p65 subunit (NF- КB) protein expression of HepG2.2.15 cells taken as control and treated with R837 (8μg/ml) for 72 h

    Article Snippet: Analysis of HBV viral properties The synthetic ligand used for TLR7 was Imiquimod-R837 provided by Invivogen.

    Techniques: Activation Assay, Expressing, Imaging