tlr4 neutralizing mouse antibody  (Novus Biologicals)

Journal: International Journal of Molecular Sciences

Article Title: BGN Secreted by Cancer-Associated Fibroblasts Promotes Esophageal Squamous Cell Carcinoma Progression via Activation of TLR4-Mediated Erk and NF-κB Signaling Pathways

doi: 10.3390/ijms262412024

Figure Lengend Snippet: Biglycan (BGN) promotes proliferation and migration of esophageal squamous cell carcinoma (ESCC) cells through its receptor TLR4. ( A ) Expression of TLR4 in ESCC cells was confirmed by Western Blotting. ( B ) Double immunofluorescence staining for BGN (green) and TLR4 (red) was performed in ESCC cells treated with recombinant human BGN (rhBGN; 100 ng/mL). Nuclei were counterstained with DAPI (blue). ( C , D ) MTS assays ( C ) and Transwell migration assays ( D ) were conducted to evaluate changes in the proliferation ( C ) and migration ( D ) of TE-9, TE-10, and TE-15 cells following treatment with rhBGN (100 ng/mL) in the presence of a TLR4-neutralizing antibody (1 μg/mL) or control immunoglobulin G (IgG; 1 μg/mL). Migrated cells were counted in five representative microscopic fields after 48 h, and representative images are shown beneath the graphs ( D ). The data are presented as the mean ± standard error of the mean (SEM) from three independent experiments ( C , D ). ** p < 0.01, *** p < 0.001. Scale bars: 20 μm ( B ) and 100 μm ( D ).

Article Snippet: Treatments applied to ESCC cells included rhBGN (#2667-CM; 100 ng/mL, R&D Systems) with or without PD98059 (Erk signaling inhibitor; 5 μM, CST), Bay 11-7082 (NF-κB signaling inhibitor: 1 μM, Sigma), DMSO vehicle (5 μM), TLR4 neutralizing mouse antibody (#NB100-56723; 1 μg/mL, Novus Biologicals, Centennial, CO, USA), or control mouse IgG (sc-2025; 1 μg/mL, Santa Cruz).

Techniques: Migration, Expressing, Western Blot, Double Immunofluorescence Staining, Recombinant, Control

Journal: International Journal of Molecular Sciences

Article Title: BGN Secreted by Cancer-Associated Fibroblasts Promotes Esophageal Squamous Cell Carcinoma Progression via Activation of TLR4-Mediated Erk and NF-κB Signaling Pathways

doi: 10.3390/ijms262412024

Figure Lengend Snippet: Biglycan (BGN) promotes proliferation, migration, and activation of macrophages and fibroblasts. ( A ) Cell–cell communication network analysis of BGN -expressing cancer-associated fibroblasts (CAFs) in esophageal squamous cell carcinoma (ESCC) tissues. Fibroblasts in single-cell RNA sequencing (scRNAseq) datasets from ESCC tissues were stratified into CAF_ BGN _High (scaled expression > 3) and CAF_ BGN _Low (≤3) groups based on BGN expression levels. Cell–cell communication inferred by CellChat revealed that CAF_ BGN _High cells exhibited strong outgoing signaling toward epithelial and myeloid cells, as well as prominent autocrine signaling within the CAF_ BGN _High population. ( B , C ) MTS assay ( B ) and Transwell migration assay ( C ) showing increased proliferation and migration of mesenchymal stem cells (MSCs) treated with recombinant human BGN (rhBGN; 100 ng/mL), respectively. ( D ) Western Blot analysis showing that fibroblast activation protein (FAP) and α-smooth muscle actin (αSMA) (CAF markers) were upregulated in MSCs treated with rhBGN for 24 h, whereas interleukin-6 (IL6) expression remained unchanged. TLR4 expression was detected; however, phosphorylation of NF-κB and Erk was not increased by rhBGN. ( E , F ) MTS assay ( E ) and Transwell migration assay ( F ) showing that rhBGN-induced proliferation and migration of MSCs were not suppressed by a TLR4-neutralizing antibody (1 μg/mL). ( G , H ) MTS assay ( G ) and Transwell migration assay ( H ) showing increased proliferation and migration of macrophages treated with rhBGN (100 ng/mL), respectively. ( I ) Western Blot analysis showing that rhBGN treatment upregulated the expression of CD163 and CD206 (M2 macrophage markers) and increased NF-κB phosphorylation, while TLR4 expression was also detected, but phosphorylated Erk was not changed. ( J , K ) MTS assay ( J ) and Transwell migration assay ( K ) showing that rhBGN-induced proliferation and migration of macrophages were attenuated by treatment with a TLR4-neutralizing antibody (1 μg/mL), respectively. ( L , M ) MTS assay ( L ) and Transwell migration assay ( M ) showing that treatment with the NF-κB pathway inhibitor Bay 11-7082 (1 μM) attenuated rhBGN-induced proliferation and migration to a similar extent, respectively. Data are presented as the mean ± standard error of the mean (SEM) from three independent experiments ( B , C , E – H , J – M ). * p < 0.05, ** p < 0.01, *** p < 0.001. N.S., not significant. Scale bars: 100 μm ( C , F , H , K , M ).

Article Snippet: Treatments applied to ESCC cells included rhBGN (#2667-CM; 100 ng/mL, R&D Systems) with or without PD98059 (Erk signaling inhibitor; 5 μM, CST), Bay 11-7082 (NF-κB signaling inhibitor: 1 μM, Sigma), DMSO vehicle (5 μM), TLR4 neutralizing mouse antibody (#NB100-56723; 1 μg/mL, Novus Biologicals, Centennial, CO, USA), or control mouse IgG (sc-2025; 1 μg/mL, Santa Cruz).

Techniques: Migration, Activation Assay, Expressing, RNA Sequencing, MTS Assay, Transwell Migration Assay, Recombinant, Western Blot, Phospho-proteomics

Journal: International Journal of Molecular Sciences

Article Title: BGN Secreted by Cancer-Associated Fibroblasts Promotes Esophageal Squamous Cell Carcinoma Progression via Activation of TLR4-Mediated Erk and NF-κB Signaling Pathways

doi: 10.3390/ijms262412024

Figure Lengend Snippet: A schematic illustration of the role of BGN in the tumor–stromal interactions among ESCC cells, CAFs, and macrophages. BGN secreted from MSCs that have undergone CAF transition upon direct contact with ESCC cells promotes ESCC cell proliferation and migration through the TLR4–Erk/NF-κB signaling pathways. In addition, BGN enhances the proliferation and migration of MSCs and induces their differentiation into CAFs. Furthermore, BGN promotes the proliferation and migration of macrophages and drives their polarization toward the M2 phenotype through the TLR4–NF-κB signaling pathways.

Article Snippet: Treatments applied to ESCC cells included rhBGN (#2667-CM; 100 ng/mL, R&D Systems) with or without PD98059 (Erk signaling inhibitor; 5 μM, CST), Bay 11-7082 (NF-κB signaling inhibitor: 1 μM, Sigma), DMSO vehicle (5 μM), TLR4 neutralizing mouse antibody (#NB100-56723; 1 μg/mL, Novus Biologicals, Centennial, CO, USA), or control mouse IgG (sc-2025; 1 μg/mL, Santa Cruz).

Techniques: Migration, Protein-Protein interactions

Journal: Frontiers in Immunology

Article Title: Toll-Like Receptor- and Protein Kinase R-Induced Type I Interferon Sustains Infection of Leishmania donovani in Macrophages

doi: 10.3389/fimmu.2022.801182

Figure Lengend Snippet: Dual role for TLR4 and TLR2 in the induction of type I IFN by L. donovani in macrophages. Total RNA was extracted from L. donovani -infected macrophages from C57BL/6, tlr2 -/- and myd88 -/- mice. Thioglycolate-recruited peritoneal macrophages were infected with stationary-phase promastigotes of L. donovani wild-type or L. donovani expressing ISP2 ( L.d:ISP 2) at a 10:1 parasite:macrophage ratio. After 2 or 3 h of infection, cultures were washed and RNA was extracted or cultures were incubated in RPMI-BSA for 6 h before extraction. Total RNA was extracted using the RNeasy kit (Qiagen). cDNA was made and used as templates in qPCR for the determination of the relative mRNA levels for IFN-β and IFN-α. Graphs show IFN-β levels in (A) C57BL/6 and tlr2 -/- macrophages from the same experiment, and in (C) C57BL/6 and myd88 -/- macrophages from the same experiment. (D) Graphs of the IFN-α levels in C57BL/6 and myd88 -/- macrophages. Where indicated, cells were pre-treated with IgG or neutralizing anti-TLR4 antibodies for 30 min prior to infection. Uninfected cells were used as a control (Ctrl). The experiment was repeated 2 independent times. The graphs show representative experiments with the means ± SD of the triplicate technical replicates. Statistical significance was assessed using one-way ANOVA with Bonferroni posttest. * p < 0.05, ** p < 0.01, *** p < 0.001. Differences between the same time point or the same group are shown. (B) Peritoneal thioglycolate-recruited macrophages from C57BL/6 and tlr2 -/- mice were cultivated on glass coverslips overnight in RPMI-FCS and then washed and infected with late-stage promastigotes of L. donovani at a 5:1 parasite:macrophage ratio for 3 h at 37°C in RPMI-BSA. After 3 h, the monolayers were washed with HBSS to remove extracellular parasites, fixed with methanol and Giemsa-stained, or further incubated for 72 h in RPMI-FCS at 37°C before fixation and staining. Where indicated, 200 ng/ml IFN-β was added to cultures that had been previously infected for 3 h and washed for removal of extracellular parasites and cultivated for 72 h in RPMI-FCS. The number of intracellular parasites was determined under a light microscope. The experiment was performed 3 independent times. The graphs show a representative experiment of the means ± SD of replicates in triplicate. Statistical significance was assessed using two-way ANOVA with Tukey’s and Sidak’s multiple comparison tests. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: In the experiments of receptor neutralization, 10 μg/ml anti-mouse TLR4 neutralizing antibodies (CD284/MD2 complex clone MTS510; Thermo Fisher Scientific) or 10 μg/ml IgG (IgG2aK control clone eBR2a; Thermo Fisher Scientific) were incubated for 30 min with the macrophages in RPMI-10% FCS and then washed twice before infection with the parasites in RPMI-0.1% BSA at 37°C and 5% CO 2 .

Techniques: Infection, Expressing, Incubation, Extraction, Control, Staining, Light Microscopy, Comparison

Journal: Frontiers in Immunology

Article Title: Toll-Like Receptor- and Protein Kinase R-Induced Type I Interferon Sustains Infection of Leishmania donovani in Macrophages

doi: 10.3389/fimmu.2022.801182

Figure Lengend Snippet: Neutrophil elastase and PKR are required for the induction of anti-inflammatory genes in infected macrophages. (A) Nuclear extracts were obtained from C57BL6 macrophages infected for 2, 6, or 24 h with stationary-phase promastigotes of L. donovani at a 10:1 parasite:macrophage ratio. The nuclear extracts were processed for Western blot analysis with antibodies to IRF3, as well as nuclear lamin for the loading control. Macrophages incubated in medium alone were used as the baseline control (Ctrl). Poly I:C was used at 25 µg/ml. In (D) , total lysates were obtained from C57BL/6 macrophages infected for 1 h with stationary-phase promastigotes of L. donovani at a 10:1 parasite:macrophage ratio and processed for Western blot with antibodies to phospho-PKR, as well as actin for the loading control. Where indicated, monolayers were pre-treated with 100 nM of the TLR4 inhibitor, TAK-242 or with 10 µM of neutrophil elastase inhibitor (NEI) before infection. Total RNA was extracted from L. donovani -infected macrophages from (B, C, E) C57BL/6 and ela2 -/- mice or (F) 129Sv/Ev and pkr -/- mice. Thioglycolate-recruited peritoneal macrophages were infected with stationary-phase promastigotes of L. donovani wild-type or L. donovani expressing ISP2 ( L.d:ISP 2) at a 10:1 parasite:macrophage ratio for 2 h, cultures were washed, and RNA was extracted or cultures were incubated in RPMI-BSA until 6 h before extraction. cDNA was made and used as a template in qPCR for the determination of the relative mRNA levels for IL10, OASL2, SOD1, and PKR as indicated. The experiment was repeated 2 independent times. The graphs show representative experiments, with the means ± SD of the triplicate technical replicates. Statistical significance was assessed using one-way ANOVA with Bonferroni posttest. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: In the experiments of receptor neutralization, 10 μg/ml anti-mouse TLR4 neutralizing antibodies (CD284/MD2 complex clone MTS510; Thermo Fisher Scientific) or 10 μg/ml IgG (IgG2aK control clone eBR2a; Thermo Fisher Scientific) were incubated for 30 min with the macrophages in RPMI-10% FCS and then washed twice before infection with the parasites in RPMI-0.1% BSA at 37°C and 5% CO 2 .

Techniques: Infection, Western Blot, Control, Incubation, Expressing, Extraction

Journal: Frontiers in Immunology

Article Title: Toll-Like Receptor- and Protein Kinase R-Induced Type I Interferon Sustains Infection of Leishmania donovani in Macrophages

doi: 10.3389/fimmu.2022.801182

Figure Lengend Snippet: Schematic representation of the role of TLRs and PKR in L. donovani development in macrophages. L. donovani utilizes macrophage NE activity, susceptible to inhibition by the ecotin-like inhibitor of serine peptidase ISP2, via TLR4 and TLR2 during phagocytosis (1), to induce PKR phosphorylation. NE contributes to the modulation of Rab7 + endosomal fusion with the phagosome (2) and to IRF3 accumulation in the nucleus (3). IRF7 recruitment to parasite-containing phagosomes takes place during early stages of infection (2). TLR3 is recruited to the parasitophorous vacuole (4), and together with integrative signals conveyed through PKR (5) acts as a hub to induce the expression of IFN-I, OASL2, SOD1, IL10, and PKR genes (6). IFNβ ensures parasite survival and growth in macrophages (7).

Article Snippet: In the experiments of receptor neutralization, 10 μg/ml anti-mouse TLR4 neutralizing antibodies (CD284/MD2 complex clone MTS510; Thermo Fisher Scientific) or 10 μg/ml IgG (IgG2aK control clone eBR2a; Thermo Fisher Scientific) were incubated for 30 min with the macrophages in RPMI-10% FCS and then washed twice before infection with the parasites in RPMI-0.1% BSA at 37°C and 5% CO 2 .

Techniques: Activity Assay, Inhibition, Phospho-proteomics, Infection, Expressing

Journal: The FASEB Journal

Article Title: Neutrophil elastase promotes Leishmania donovani infection via interferon-β

doi: 10.1096/fj.201900524R

Figure Lengend Snippet: TLR4 and TLR2 are required for development of L. donovani in macrophages, and NE acts through TLR4. Peritoneal thioglycolate-recruited macrophages from C57BL/6 mice, TLR2 knockout mice ( tlr 2 −/− ), or TLR4 knockout mice ( tlr 4 −/− ) ( A ) or from NE knockout mice ( ela 2 −/− ) ( B ) were cultivated on glass coverslips overnight in RPMI-FCS and washed and infected with late-stage promastigotes of L. donovani at a 5:1 parasite:macrophage ratio for 3 h at 37°C in RPMI supplemented with 0.1% BSA. The monolayers were washed 3 times to remove extracellular parasites, fixed with methanol and Giemsa stained (3 h), or further cultivated for 72 h in RPMI-FCS at 37°C ( A ). NE acts through TLR4. B ) Where indicated, macrophages were pretreated with 10 μg/ml of control IgG2b or with anti-TLR4 (MTS5) mAb for 30 min in RPMI-FCS and washed before infection. Where indicated (+), purified human NE was added at 200 ng/ml after removal of antibodies and before addition of the parasites. The experiments were performed in triplicate and repeated at least 3 independent times. The graphs show 1 representative experiment. The graphs show means ± sd . * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: In the experiments of receptor neutralization, 10 μg/ml anti-mouse TLR4 neutralizing antibodies (CD284/MD2 complex clone MTS510; Thermo Fisher Scientific) or 10 μg/ml IgG (IgG2aK control clone eBR2a) were incubated for 30 min with macrophages in RPMI-FCS, and the monolayers were washed twice before infection with the parasites at 37°C in RPMI-BSA.

Techniques: Knock-Out, Infection, Staining, Control, Purification

Journal: The FASEB Journal

Article Title: Neutrophil elastase promotes Leishmania donovani infection via interferon-β

doi: 10.1096/fj.201900524R

Figure Lengend Snippet: Colocalization of NE and TLR4 with L. donovani in infected macrophages. Macrophages of C57BL/6 mice were infected with WT L. donovani or L. donovani : ISP 2 (clone 2) for 3 h in RPMI-BSA and processed for immunofluorescence or washed and cultured for 72 h in RPMI-FCS and processed for immunofluorescence. Coverslips were treated with anti-TLR4 antibodies washed and incubated with anti-rabbit–Alexa 488. Coverslips were subsequently incubated with antibodies to NE and washed and incubated with anti-rabbit–cyanine 3 antibodies. Coverslips were treated with DAPI for 5 min and washed and mounted for immunofluorescence. Samples were analyzed by confocal microscopy. White arrows point to the parasite. Scale bar, 2 μm.

Article Snippet: In the experiments of receptor neutralization, 10 μg/ml anti-mouse TLR4 neutralizing antibodies (CD284/MD2 complex clone MTS510; Thermo Fisher Scientific) or 10 μg/ml IgG (IgG2aK control clone eBR2a) were incubated for 30 min with macrophages in RPMI-FCS, and the monolayers were washed twice before infection with the parasites at 37°C in RPMI-BSA.

Techniques: Infection, Immunofluorescence, Cell Culture, Incubation, Confocal Microscopy

Journal: Toxins

Article Title: An Extract of Rhodobacter sphaeroides Reduces Cisplatin-Induced Nephrotoxicity in Mice

doi: 10.3390/toxins5122353

Figure Lengend Snippet: Lycogen™ reduced cisplatin-induced cell apoptosis. MES-13 cells were pretreated with Lycogen™ at concentration of 0, 2, 4, 8 or 16 μM for 48 h. Next, cisplatin (5 μg/mL) was added to the cells for 48 h. ( A ) Cell viability was measured using a WST-1 assay; ( B ) The expression of nuclear p53 and caspase 3 was measured by Western blot analysis. Proliferating cell nuclear antigen (PCNA) and β-actin expression served as loading controls for nuclear proteins and total proteins, respectively. Inserted values indicate the relative protein expression when compared with pro-caspase 3. * p < 0.05, ** p < 0.01, *** p < 0.001 (mean ± SD, n = 6); ( C ) TNF-α secreted by MES-13 cells is mediated by TLR4 signaling. The cells were preincubated with anti-TLR4 or with control IgG, and then treated with or without Lycogen™ for 48 h. TNF-α was measured using an enzyme-linked immunosorbent assay. Each experiment was repeated three times with similar results.

Article Snippet: The MES-13 cells were preincubated with 10 μg of neutralizing antibody against mouse TLR4 (MTS510, eBioscience, San Diego, CA, USA) or with control IgG (eBioscience) for 30 min at 25 °C, and then treated with LycogenTM or cisplatin in the culture medium for 48 h. The level of TNF-α in the supernatant was determined by ELISA (R&D).

Techniques: Concentration Assay, WST-1 Assay, Expressing, Western Blot, Control, Enzyme-linked Immunosorbent Assay