Journal: Nature immunology

Article Title: Genetic dissection of TLR9 reveals complex regulatory and cryptic proinflammatory roles in mouse lupus.

doi: 10.1038/s41590-022-01310-2

Figure Lengend Snippet: Fig. 4 | Effects of TLR9 and sex on TLR7 expression and distribution. a–d, Intracellular staining of TLR7 and TLR9 (ratio of TLR7 MFI over average TLR7 MFI of the corresponding male or female WT group) in FO (a,c) and MZ (b,d) B cells of Tlr9-mutant male or female BALB/c mice. Data points indicate individual mice (Tlr9K51E/K51E male, n = 7; female, n = 11; Tlr9 WT male, n = 7, female, n = 11; Tlr9P915H/P915H male, n = 7, female, n = 10; Tlr9+/− male, n = 3, female, n = 7; Tlr9−/− male, n = 7, female, n = 11), and bars indicate the mean ± s.e.m. of two or three experiments pooled (except from one experiment for Tlr9+/− males); *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001, one-way ANOVA with Tukey’s multiple comparisons test; female, Tlr9K51E/K51E versus Tlr9−/−, P = 0.0026; Tlr9−/− versus Tlr9P915H/P915H, P = 0.0251; Tlr9WT versus Tlr9−/−, P = 0.0003 (a); female, Tlr9K51E/K51E versus Tlr9−/−, P = 0.0170; Tlr9WT versus Tlr9P915H/

Article Snippet: Cells were stained at 50–100 million cells per ml overnight at 4 °C in 1× Perm/Wash buffer with fluorochrome-conjugated antibodies CD45R (clone RA3-6B2, 1:400), TLR7 (clone A94B10, 1:800), TLR9–biotin (clone Nar9, from K. Miyake67, biotin conjugation in-house, 1:600) and unconjugated EEA1 (polyclonal anti-rabbit, Novus, 1:100) or LAMP-1 (polyclonal anti-rabbit, Abcam, 1:100).

Techniques: Expressing, Staining, Mutagenesis

Journal: PLoS ONE

Article Title: Cholecalciferol decreases inflammation and improves vitamin D regulatory enzymes in lymphocytes in the uremic environment: A randomized controlled pilot trial

doi: 10.1371/journal.pone.0179540

Figure Lengend Snippet: Expression of TLR7, TLR9, IL-6, IFN-γ, VDR, CYP27b1 and CYP24a1 in B and T lymphocytes.

Article Snippet: After lysing, cells were permeabilized as described above to intracellular stain with 5 μL pf FITC-labeled IL-6 (BD Biosciences, San Diego, USA), 10 μL of PE-labeled TLR7 antibody (R&D Systems, Mineapolis, USA), 5 μL of APC-Cy7-labeled IFN-γ (eBiosciences, San Diego, USA) and 10 μL of APC-labeled TLR9 (BD Biosciences, San Diego, USA) antibodies.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Cholecalciferol decreases inflammation and improves vitamin D regulatory enzymes in lymphocytes in the uremic environment: A randomized controlled pilot trial

doi: 10.1371/journal.pone.0179540

Figure Lengend Snippet: Effect of 25 and 1,25 vitamin D on TLR7 and TLR9 expression (MFI) in B lymphocytes (Figs A and B) and T lymphocytes (Figs C and D) in presence of healthy or uremic serum (US) and after CYP24 silencing (siRNA).

Article Snippet: After lysing, cells were permeabilized as described above to intracellular stain with 5 μL pf FITC-labeled IL-6 (BD Biosciences, San Diego, USA), 10 μL of PE-labeled TLR7 antibody (R&D Systems, Mineapolis, USA), 5 μL of APC-Cy7-labeled IFN-γ (eBiosciences, San Diego, USA) and 10 μL of APC-labeled TLR9 (BD Biosciences, San Diego, USA) antibodies.

Techniques: Expressing

Journal: BMC Complementary Medicine and Therapies

Article Title: Huang Lian Jie Du Decoction enhances the anti-tumor efficacy of immune checkpoint inhibitors by activating TLR7/8 signalling in melanoma

doi: 10.1186/s12906-024-04444-y

Figure Lengend Snippet: Toll-like receptor signaling pathway is activated after HLJD treatment. ( A ) RNA-seq analysis was performed and the volcano plot was demonstrated. ( n = 3 mice/group) ( B ) Interaction net of the significant pathways (Path-Net) of differentially signaling pathways. ( C ) Immunohistochemistry was used to analyze the expression levels of TLR7 (400×). HLJD = Huang Lian Jie Du Decoction, ICIs = anti-PD-1 + anti-CTLA-4

Article Snippet: Primary antibody against mouse TLR7 was purchased from Proteintech (Wuhan, China), and secondary antibodies and DAPI were obtained from Servicebio (Wuhan, China).

Techniques: RNA Sequencing, Protein-Protein interactions, Immunohistochemistry, Expressing

Journal: BMC Complementary Medicine and Therapies

Article Title: Huang Lian Jie Du Decoction enhances the anti-tumor efficacy of immune checkpoint inhibitors by activating TLR7/8 signalling in melanoma

doi: 10.1186/s12906-024-04444-y

Figure Lengend Snippet: HLJD treatment upregulated Type I IFN signaling. ( A , B and C ) Volcano Plot graph and Bar graphs of PCR-array of TLR7/8 and Type I IFN signaling axis. ( D ) The expression of IRF7 was determined by qRT-PCR. ( E ) IFN-α quantification by ELISA in tumor tissue. Statistical differences were determined using the student’s t-test. (* p < 0.05, ** p < 0.01) HLJD = Huang Lian Jie Du Decoction, ICIs = anti-PD-1 + anti-CTLA-4

Article Snippet: Primary antibody against mouse TLR7 was purchased from Proteintech (Wuhan, China), and secondary antibodies and DAPI were obtained from Servicebio (Wuhan, China).

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

Journal: Inflammation Research

Article Title: TLR7 activation in epilepsy of tuberous sclerosis complex

doi: 10.1007/s00011-019-01283-3

Figure Lengend Snippet: TLR7 transcript and protein are expressed in TSC cortex. a TLR7 transcript expression is present in all TSC specimens. Expression of TLRs 2, 4 and 7 was measured using the NanoString nCounter neuroinflammation assay and values for each specimen were normalized to the mean for the given TLR and shown in log2 scale. TLR7 was also quantified using qPCR. Expression levels were concordant between the two technologies ( P = 0.0162, Spearman’s rho). Levels for the TLRs were not statistically significant when compared between categories. b Protein levels of signaling-competent TLR7 were detected in membrane fractions. Capillary electrophoresis Western analysis of membrane (M) and cytosolic (C) fractions was performed on two specimens for each tissue category. Calnexin was used as a marker specific to membrane fractions. Results are displayed as pseudo blots

Article Snippet: Antibodies for TLR7 (Santa Cruz SC-57463) and calnexin (RayBiotech) were used.

Techniques: Expressing, Membrane, Electrophoresis, Western Blot, Marker

Journal: Inflammation Research

Article Title: TLR7 activation in epilepsy of tuberous sclerosis complex

doi: 10.1007/s00011-019-01283-3

Figure Lengend Snippet: Evidence of TLR7 pathway activation in AMT-hot epileptogenic TSC tubers. a IL-23A expression is an established marker of TLR7 activation. Using qPCR we measured expression of IL-23A in each category. Two-way ANOVA was used to investigate the relationship of IL-23A to AMT uptake and seizure onset status. IL-23A is significantly associated with AMT uptake and increased in AMT-hot tubers (OH), P = 0.0387, F ratio = 5.206. 95% confidence intervals (diamonds) are shown for AMT hot and cold groups, with mean values and overlap marks (horizontal bars near the tips of the diamonds). Vertical separation between the overlap marks of the two diamonds indicates statistical significance. N = 4,5,4,4, respectively, for normal, NC, OC, and OH categories. b Regression analysis demonstrates that expression of miR-142-3p (a microRNA ligand and activator of TLR7) is a significant predictor of IL-23A mRNA levels in TSC tissue ( P = 0.0318). c A set of proteins characteristic for TLR7 activation is significantly increased in OH tubers ( P = 0.0283). Proteins established as a signature of TLR7 activation were analyzed using our previous quantitative proteomics dataset comparing OH vs normal control (NT) . MS/MS spectra were available for nine signature proteins (C1QB, TRAFD1, HSPH1, TNS3, TAPBP, PSMB9, TAP2, TRADD, TNFAIP2). The difference in expression for each protein was calculated as log(OH/NT). Positive values above the dashed line show increased expression for 8 of 9 proteins in OH specimens. d The median level of MyD88 protein is increased in OH tubers, albeit above statistical significance, P = 0.0665. e MAPK1 protein, downstream of TLR7, is increased in OH tubers, P = 0.0006. f A set of proteins known to be induced by NF-kB is significantly increased in OH tubers, P < 0.0001. This observation was originally reported in . Here, we present the change in expression for each NF-kB target protein, calculated as log(OH/NT). Positive values above the dashed line indicate increased expression for 20 of 24 NF-kB target proteins in OH specimens

Article Snippet: Antibodies for TLR7 (Santa Cruz SC-57463) and calnexin (RayBiotech) were used.

Techniques: Activation Assay, Expressing, Marker, Quantitative Proteomics, Control, Tandem Mass Spectroscopy

Journal: Scientific Reports

Article Title: Vitamin D Deficiency Promotes Liver Tumor Growth in Transforming Growth Factor-β/Smad3-Deficient Mice Through Wnt and Toll-like Receptor 7 Pathway Modulation

doi: 10.1038/srep30217

Figure Lengend Snippet: ( a ) Total mRNA was isolated from liver tumors of both wild type and Smad3 +/− mice which were fed either low VD or high VD regimen (n = 3 for each group). Gene expression levels of TLR7, TLR9, OAZ1, and P-Para were measured by Q-PCR. Each result shown is representative of three independent experiments. Error bars are shown as standard deviations. (*p < 0.05 [WT Low VD vs. Smad3 +/− Low VD], **p < 0.05 [Smad3 +/− Low VD vs. Smad3 +/− High VD], Student’s t-test). ( b ) A schematic representation of the TLR7 promoter studied by ChIP analysis. Two sets of PCR primers, designated as P1 and P2, were used for amplification of the TLR7 promoter. SBE (Smad-binding element) positions are numbered relative to the major transcription start site (+1). Black arrow indicates transcription start site. ( c ) HepG2 cells were treated with 200 pM TGF-β for 2 hrs. ChIP experiments were performed as described in Methods. ( d ) Gaussia luciferase plasmid containing a TLR7 promoter region was transfected into HepG2 shRNA-Control and HepG2 shRNA-SMAD3 cells. The secondary reporter, secreted Alkaline Phosphatase, serves as an internal control. TLR7 transcriptional activity was analyzed after 200 pM TGF-β and 100 nM VD treatment for 24 hrs. Error bars are shown as SD in ( c , d ). Each result shown is representative of three independent experiments (*p < 0.01, **p < 0.001, Student’s t-test). ( e ) Knockdown TLR7 suppresses HepG2 cell growth and cell migration. Cell proliferation of HepG2-siCtrl and HepG2-siTLR7 cells were assessed by colorimetric MTS assays. Transwell migration assays of HepG2-siCtrl and HepG2-siTLR7 cells were performed. Cells were treated with TGF-β1 (200 pM) for 24 hours. HepG2 cells were transfected with control-siRNA or TLR7-siRNA. The expression of TLR in HepG2-siCtrl and HepG2-siTLR7 cells were measured by Q-PCR analyses. Results are presented as mean ± SD (*p < 0.05, **p < 0.01, Student’s t-test).

Article Snippet: Lentiviral particles containing shRNA to Smad3 (sc-38376), control shRNA (sc-108080), siRNA to TLR7 (sc-40266) or control siRNA (sc-44231) were purchased from Santa Cruz Biotechnology and were used to infect HepG2 cells. siRNA for β-catenin (L-003448-00-0005) and siRNA control Non-targeting Pool (# D-001810-10-05) were purchased from Dharmacon (Pittsburgh PA).

Techniques: Isolation, Gene Expression, Amplification, Binding Assay, Luciferase, Plasmid Preparation, Transfection, shRNA, Control, Activity Assay, Knockdown, Migration, Expressing

Journal: International immunology

Article Title: Cross-talk among Toll-like receptors and their ligands.

doi: 10.1093/intimm/dxn027

Figure Lengend Snippet: Fig. 1. Western blot analysis of TLR expression in STC-1 cells. The murine enteroendocrine cell line STC-1 was treated with TLR ligands LPS (1 lg ml1), CpG-ODN 1668 (1 lM), flagellin (Flag, 100 ng ml1), loxiribine (Lox, 10 lM) and PGN (10 lg ml1) or left untreated (Untr). Protein expression levels of TLR4, TLR5, TLR7 and TLR9 were analysed by western blot analysis (A), followed by scanning densitometry (B–E). Beta-actin protein expression was used as a reference loading control. A significant image of three different experiments performed in triplicate is shown.

Article Snippet: Antibodies used for western blot analysis were as follows: goat anti-mouse TLR4 cross-reacting with human TLR4 (L-14, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), rabbit anti-mouse TLR5 cross-reacting with human TLR5 (H-127, Santa Cruz Biotechnology, Inc), rabbit anti-human TLR7 crossreacting with mouse TLR7 (IMG-581, Imgenex, San Diego, CA, USA), biotinylated mouse anti-human TLR9 cross-reacting with mouse TLR9 (HBT, Uden, The Netherlands) and mouse mAb to beta-actin cross-reacting with human (AC15, Abcam, Cambridge, UK).

Techniques: Western Blot, Expressing, Control

Journal: International immunology

Article Title: Cross-talk among Toll-like receptors and their ligands.

doi: 10.1093/intimm/dxn027

Figure Lengend Snippet: Fig. 2. Western blot analysis of TLR expression in LCC-18 cells. The human enteroendocrine cell line LCC-18 was treated with TLR ligands LPS (1 lg ml1), CpG-ODN 2006 (1 lM), flagellin (Flag, 100 ng ml1), loxiribine (Lox, 10 lM) and PGN (10 lg ml1) or left untreated (Untr). Protein expression levels of TLR4, TLR5, TLR7 and TLR9 were analysed by western blot analysis (A), followed by scanning densitometry (B–E). Beta- actin protein expression was used as a reference loading control. A significant image of three different experiments performed in triplicate is shown.

Article Snippet: Antibodies used for western blot analysis were as follows: goat anti-mouse TLR4 cross-reacting with human TLR4 (L-14, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), rabbit anti-mouse TLR5 cross-reacting with human TLR5 (H-127, Santa Cruz Biotechnology, Inc), rabbit anti-human TLR7 crossreacting with mouse TLR7 (IMG-581, Imgenex, San Diego, CA, USA), biotinylated mouse anti-human TLR9 cross-reacting with mouse TLR9 (HBT, Uden, The Netherlands) and mouse mAb to beta-actin cross-reacting with human (AC15, Abcam, Cambridge, UK).

Techniques: Western Blot, Expressing, Control

Journal: International immunology

Article Title: Cross-talk among Toll-like receptors and their ligands.

doi: 10.1093/intimm/dxn027

Figure Lengend Snippet: Fig. 3. Western blot analysis of TLR expression in HT29 cells. The human enterocyte cell line HT-29 was treated with TLR ligands LPS (1 lg ml1), CpG-ODN 2006 (1 lM), flagellin (Flag, 100 ng ml1), loxiribine (Lox, 10 lM) and PGN (10 lg ml1) or left untreated (Untr). Protein expression levels of TLR4, TLR5, TLR7 and TLR9 were analysed by western blot analysis (A), followed by scanning densitometry (B–E). Beta-actin protein expression was used as a reference loading control. A significant image of three different experiments performed in triplicate is shown.

Article Snippet: Antibodies used for western blot analysis were as follows: goat anti-mouse TLR4 cross-reacting with human TLR4 (L-14, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), rabbit anti-mouse TLR5 cross-reacting with human TLR5 (H-127, Santa Cruz Biotechnology, Inc), rabbit anti-human TLR7 crossreacting with mouse TLR7 (IMG-581, Imgenex, San Diego, CA, USA), biotinylated mouse anti-human TLR9 cross-reacting with mouse TLR9 (HBT, Uden, The Netherlands) and mouse mAb to beta-actin cross-reacting with human (AC15, Abcam, Cambridge, UK).

Techniques: Western Blot, Expressing, Control

Journal: International immunology

Article Title: Cross-talk among Toll-like receptors and their ligands.

doi: 10.1093/intimm/dxn027

Figure Lengend Snippet: Fig. 4. Western blot analysis of TLR expression in RAW 264.7 cells. The murine macrophagic cell line RAW 264.7 was treated with TLR ligands LPS (1 lg ml1), CpG-ODN 1668 (1 lM), flagellin (Flag, 100 ng ml1), loxiribine (Lox, 10 lM) and PGN (10 lg ml1) or left untreated (Untr). Protein expression levels of TLR4, TLR5, TLR7 and TLR9 were analysed by western blot analysis (A), followed by scanning densitometry (B–E). Beta-actin protein expression was used as a reference loading control. A significant image of three different experiments performed in triplicate is shown.

Article Snippet: Antibodies used for western blot analysis were as follows: goat anti-mouse TLR4 cross-reacting with human TLR4 (L-14, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), rabbit anti-mouse TLR5 cross-reacting with human TLR5 (H-127, Santa Cruz Biotechnology, Inc), rabbit anti-human TLR7 crossreacting with mouse TLR7 (IMG-581, Imgenex, San Diego, CA, USA), biotinylated mouse anti-human TLR9 cross-reacting with mouse TLR9 (HBT, Uden, The Netherlands) and mouse mAb to beta-actin cross-reacting with human (AC15, Abcam, Cambridge, UK).

Techniques: Western Blot, Expressing, Control

Journal: International immunology

Article Title: Cross-talk among Toll-like receptors and their ligands.

doi: 10.1093/intimm/dxn027

Figure Lengend Snippet: Fig. 5. PCR analysis of TLR expression in different cell lines. The STC-1, LCC-18, HT29 and RAW 264.7 cell lines were treated with TLR ligands LPS (1 lg ml1), CpG-ODN 2006 (1 lM), flagellin (Flag, 100 ng ml1), loxiribine (Lox, 10 lM) and PGN (10 lg ml1) or left untreated (Untr). mRNA expression levels of TLR4, TLR5, TLR7 and TLR9 were analysed by PCR analysis. 1, untreated cells; 2, LPS; 3, CpG-ODN; 4, flagellin; 5, loxiribine and 6, PGN.

Article Snippet: Antibodies used for western blot analysis were as follows: goat anti-mouse TLR4 cross-reacting with human TLR4 (L-14, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), rabbit anti-mouse TLR5 cross-reacting with human TLR5 (H-127, Santa Cruz Biotechnology, Inc), rabbit anti-human TLR7 crossreacting with mouse TLR7 (IMG-581, Imgenex, San Diego, CA, USA), biotinylated mouse anti-human TLR9 cross-reacting with mouse TLR9 (HBT, Uden, The Netherlands) and mouse mAb to beta-actin cross-reacting with human (AC15, Abcam, Cambridge, UK).

Techniques: Expressing