mouse anti-human tlr4-pe clone: hta125  (Thermo Fisher)

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: Antioxidants

Article Title: In Vitro Effects of Lipopolysaccharide on Rabbit Sperm: Toll-like Receptor 4 Expression, Motility, and Oxidative Status

doi: 10.3390/antiox14040431

Figure Lengend Snippet: UV (ultraviolet) micrographs of rabbit spermatozoa without ( A ) and after 4 h of incubation with 400 μg/mL LPS (lipopolysaccharide) ( B ) treated with anti-TLR4 antibody. The label is localized in the middle piece of the sperm tail. In ( A ), the TLR4 (Toll-like receptor 4) signal was weak, whereas in ( B ), after LPS incubation, an intense TLR4 signal was observed. Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole). Scale bars: 5 μm.

Article Snippet: Then, the slides were incubated overnight at 4 °C in a humid chamber with the primary antibodies: CD284 (TLR4) Monoclonal Antibody (HTA125) eBioscence, (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA) diluted 1:200.

Techniques: Incubation, Staining