mouse monoclonal anti tlr4 antibody  (Cell Signaling Technology Inc)

Journal: Frontiers in Immunology

Article Title: Extracellular caspase-1: a critical inducer and a therapeutic target of lung injury in gut ischemia-reperfusion

doi: 10.3389/fimmu.2026.1811868

Figure Lengend Snippet: eCasp-1 binds to TLR4 to drive inflammation, which is effectively suppressed by the novel peptide C16. (A, B) Computational modeling predicted a strong interaction between eCasp-1 (red) and TLR4 (blue). (C) SPR analysis on the binding of eCasp-1 to TLR4 in vitro . (D, E) WT and TLR4 -/- peritoneal macrophages were treated with PBS or eCasp-1 (0.1 µg/ml) for 4 h. (D) IL-6 and (E) TNFα levels in the supernatants were measured by ELISA. (F, G) WT and TLR4 -/- mice received i.p. injections of PBS or eCasp-1 (5 µg/g BW), and plasma was collected 24 h later to measure (F) IL-6 and (G) TNFα. (H) In-silico analysis identified a putative binding site for mouse eCasp-1 (red) on the extracellular domain of TLR4 (blue). (I) C16 (silver), a 16-amino-acid peptide mimic, was designed based on the predicted binding interface and exhibited strong binding to eCasp-1 (red). (J) Computational modeling predicted a potential interaction between the eCasp-1-C16 complex and TLR4. (K) SPR analysis of eCasp-1 binding to TLR4 in the presence or absence of C16. (L) WT peritoneal macrophages were treated with eCasp-1 (0.1 µg/mL) with increasing concentrations of C16 (0.1, 1, 10 µg/mL) for 4 h, and TNFα levels in the supernatants were measured by ELISA. Experiments were repeated 2–3 times and all the data obtained were used for analysis. Data were expressed as mean ± SEM (n = 5–9 samples/group) and compared by one-way analysis of variance and Student-Newman-Keuls method ( * p < 0.05 vs. WT PBS; # p < 0.05 vs. WT with eCasp-1, (+)eCasp-1 (-)C16).

Article Snippet: Recombinant mouse TLR4 (rmTLR4; ≥90% purity) was purchased from R&D Systems (Cat. No. 9149-TR-050, Minneapolis, MN), supplied in carrier-free form, and reconstituted in sterile PBS according to the manufacturer’s instructions.

Techniques: Binding Assay, In Vitro, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, In Silico

Journal: Frontiers in Immunology

Article Title: Extracellular caspase-1: a critical inducer and a therapeutic target of lung injury in gut ischemia-reperfusion

doi: 10.3389/fimmu.2026.1811868

Figure Lengend Snippet: Summary of findings. In gut I/R injury, inflammasomes activation promotes the cleavage of caspase-1 and the extracellular release of its p20 subunit through GSDMD-dependent membrane processes. This release may occur in association with GSDMD pore formation as well as membrane disruption during lytic cell death (pyroptosis). Once released, extracellular caspase-1 (eCasp-1) acts as a potent DAMP by binding to TLR4, thereby amplifying release of inflammatory cytokines, aggravating lung injury. Therapeutic intervention with the inhibitory peptide C16, which specifically blocks the eCasp-1-TLR4 interaction, effectively attenuates systemic inflammation and improves survival outcomes. I/R, Ischemia-reperfusion; GSDMD, Gasdermin-D; eCasp-1, Extracellular caspase-1; DAMP, Damage-associated molecular pattern; TLR4, Toll-like receptor 4.

Article Snippet: Recombinant mouse TLR4 (rmTLR4; ≥90% purity) was purchased from R&D Systems (Cat. No. 9149-TR-050, Minneapolis, MN), supplied in carrier-free form, and reconstituted in sterile PBS according to the manufacturer’s instructions.

Techniques: Activation Assay, Membrane, Disruption, Binding Assay

Journal: Frontiers in Immunology

Article Title: Nocardia rubra cell wall skeleton-induced MARCO expression: implications for improved phagocytosis and cytokine secretion in tumor-associated macrophages

doi: 10.3389/fimmu.2026.1611476

Figure Lengend Snippet: Nr-CWS affects MARCO expression through TLR4. The expression of TLR4 significantly increased after Nr-CWS treatment [ (D-G) , scale bars = 100 μm], while the expression TLR2 was unchanged (A-C) . Subsequent inhibition of TLR4 expression resulted in decreased levels of MARCO [ (H-J) , scale bars = 100 μm]. n = 3, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The following commercial antibodies (vendor, catalog number and dilution) utilized for western blot and immunohistochemical staining were employed in accordance with the manufacturers’ guidelines: mouse anti-CD68 antibody (Abcam, ab201340, 1:200), rabbit anti-MARCO antibody (Abcam, ab231046, 1:1000 for western blot and 1:100 for immunohistochemistry), rabbit anti-CD163 antibody (Abways Technology, CY6845, 1:500), mouse anti-CD86 antibody (Proteintech, 68674-2-Ig, 1:5000), mouse anti-TLR4 antibody (Santa Cruz Biotechnology, sc-293072, 1:1000 for western blot and 1:200 for immunohistochemistry), rabbit anti-TLR2 antibody(Abways Technology, CY5102, 1:2000), mouse anti-GAPDH antibody (Proteintech, 6004-1-Ig, 1:50000), HRP-goat anti-mouse recombinant secondary antibody (H+L)(Proteintech, RGAM001, 1:5000), goat anti-mouse IgG (H+L) Alexa Fluor 594 (Abways Technology, AB0152, 1:300), HRP-goat anti-rabbit recombinant secondary antibody (H+L) (Proteintech, RGAR001, 1:5000), goat anti-rabbit IgG (H+L) secondary antibody DyLightTM 594 (Report Biotech, S7002, 1:500), goat anti-mouse IgG (H+L) secondary antibody DyLightTM 488 (Report Biotech, S6001, 1:500), goat anti-mouse IgG (H+L) secondary antibody DyLightTM 594 (Abways Technology, AB0152, 1:500).

Techniques: Expressing, Inhibition

Journal: Frontiers in Immunology

Article Title: Electroacupuncture alleviates pain by activating the MD2/TLR4/NF-κB pathway in the ST36 acupoint

doi: 10.3389/fimmu.2025.1626755

Figure Lengend Snippet: EA-induced analgesia is associated with MD2, TLR4, and p-p65/p65 in the ST36 acupoint and MD2 and TLR4 interactions. (A) The experimental protocol timeline is illustrated, with the duration of each intervention clearly indicated in the figure. (B) The location of the ST36 acupoint in mice (up) and the swelling of paw comparison before (right) and after (left) CFA injection. (C) PTWTs were detected on the D-3 to D-1, D1pre, D1post, D3, D5, and D7 after interventions (n = 8), ** P < 0.01 vs. the CON, ※※ P < 0.01 vs. the SHAM. (D) The expression levels of PKC protein in the spinal cord were determined by WB (n = 3), ** P < 0.01 vs. the CON, ** P < 0.01 vs. the SHAM. (E, F) The expression levels of CX3CL1 (E) , IL-1β (F) in the spinal cord were determined by Elisa (n = 6), ** P < 0.01 vs. the CON, ** P < 0.01 vs. the SHAM. (G) Data sourced from https://cn.string-db.org/ . (H) Co-IP assay with TLR4 antibody (or IgG) in the ST36 acupoint, followed by WB analysis of MD2 and TLR4. (I–K) The expression levels of MD2 (I) , TLR4 (J) , and p-p65/p65 (K) were determined using WB analysis in the ST36 acupoint on D7 (n = 3), * P < 0.05 vs. the CON, ** P < 0.01 vs. the CON, ※※ P < 0.01 vs. the SHAM.

Article Snippet: Tissue lysate (1 mg protein) was incubated with either 2 μg of mouse monoclonal anti-TLR4 antibody or 2 μg of mouse IgG isotype control antibody (5415, Cell Signaling Technology, USA) for 1 hour at room temperature.

Techniques: Comparison, Injection, Expressing, Enzyme-linked Immunosorbent Assay, Co-Immunoprecipitation Assay

Journal: Frontiers in Immunology

Article Title: Electroacupuncture alleviates pain by activating the MD2/TLR4/NF-κB pathway in the ST36 acupoint

doi: 10.3389/fimmu.2025.1626755

Figure Lengend Snippet: The effects of modulating MD2 in the ST36 acupoint on EA-induced analgesia and on the protein levels of MD2, TLR4, and p-p65/p65. (A) Lentivirus injection, behavioral testing, and acupoint stimulation experimental schedule. (B) Detection of lentivirus transduction in the ST36 acupoint by IF assay after MD2 knockdown lentivirus injection (n=3). (C) IF assay detection of lentivirus transduction in ST36 acupoint after overexpression lentivirus injection (n=3). (D) The transfection efficiency of the control lentivirus (FR-376) in ST36 acupoint (n=3). (E) The transfection efficiency of the control lentivirus (FV-115) in ST36 acupoint (n=3). (F) The expression of MD2 after injection of three different target knockdown lentiviruses were detected by WB (n=4). (G) The expression of MD2 after injection of overexpression was detected by WB (n=3). (H) The PTWTs were detected on D-3 to D-1, D1pre, D1post to D7 (every other day) (n = 6), ** P < 0.01 vs. the AIA+LVcon, # P < 0.05 vs. the AIA+LVcon+EA, ## P < 0.01 vs. the AIA+LVcon+EA. (I–K) The expression level of MD2 (I) , TLR4 (J) , and p-p65/p65 (K) (n = 3), ** P < 0.01 vs. the AIA+LVcon, # P < 0.05 vs. the AIA+LVcon+EA, ## P < 0.01 vs. the AIA+LVcon+EA. (L) PTWTs were detected on D-3 to D-1, D1pre, D1post to D7 (every other day) (n = 6), * P < 0.05 vs. the AIA+OEcon, ** P < 0.01 vs. the AIA+OEcon. (M–O) The expression level of MD2 (M) , TLR4 (N) , and p-p65/p65 (O) (n = 3), ** P < 0.01 vs. the AIA+OEcon.

Article Snippet: Tissue lysate (1 mg protein) was incubated with either 2 μg of mouse monoclonal anti-TLR4 antibody or 2 μg of mouse IgG isotype control antibody (5415, Cell Signaling Technology, USA) for 1 hour at room temperature.

Techniques: Injection, Transduction, Knockdown, Over Expression, Transfection, Control, Expressing

Journal: Frontiers in Immunology

Article Title: Electroacupuncture alleviates pain by activating the MD2/TLR4/NF-κB pathway in the ST36 acupoint

doi: 10.3389/fimmu.2025.1626755

Figure Lengend Snippet: EA treatment can increase the co-expression of MD2/TLR4, TLR4-Vimentin, TLR4-Tryptase, as well as TLR4-F4/80 at the ST36 acupoint. (A) Double-labeling immunofluorescence of TLR4 (green), MD2 (red) and DAPI (blue), scale bar = 20 µm. (B) Double-labeling immunofluorescence of TLR4 (green), fibroblasts (red) and DAPI (blue), scale bar = 20 µm. (C) Double-labeling immunofluorescence of TLR4 (green) and Tryptase (red), scale bar = 20µm. (D) Double-labeling immunofluorescence of TLR4 (green) and F4/80 (red), DAPI (blue), scale bar = 20µm. (E) Immunofluorescence quantitative analysis of MD2/TLR4 (n = 3), * P < 0.05 vs. the CON, ## P < 0.01 vs. the AIA. (F) Immunofluorescence quantitative analysis of TLR4-Vimentin (n = 3), ** P < 0.01 vs. the CON, ## P < 0.01 vs. the AIA. (G) Immunofluorescence quantitative analysis of TLR4-Tryptase (n = 3), # P < 0.05 vs. the AIA. (H) Immunofluorescence quantitative analysis of TLR4-F4/80 (n = 3).

Article Snippet: Tissue lysate (1 mg protein) was incubated with either 2 μg of mouse monoclonal anti-TLR4 antibody or 2 μg of mouse IgG isotype control antibody (5415, Cell Signaling Technology, USA) for 1 hour at room temperature.

Techniques: Expressing, Labeling, Immunofluorescence

Journal: Frontiers in Immunology

Article Title: Electroacupuncture alleviates pain by activating the MD2/TLR4/NF-κB pathway in the ST36 acupoint

doi: 10.3389/fimmu.2025.1626755

Figure Lengend Snippet: The pattern of the effect of EA treatment on MD2, TLR4 in ST36 acupoint. (A) Behavioral testing, acupoint stimulation and molecular detection experimental schedule. (B–D) PTWTs were detected on D1post (B) (n=8), D3 (C) (n=6), and D5 (D) (n=6), ** P < 0.01 vs. the CON, ※ P < 0.05 vs. the SHAM, ※※ P < 0.01 vs. the SHAM. (E–H) The expression level of MD2 in the ST36 acupoint was determined by WB on D1post (E) , D3 (F) and D5 (G) (n = 3); the TLR4 was determined on the D5 (H) (n = 3), * P < 0.05 vs. the CON, ** P < 0.01 vs. the CON, # P < 0.05 vs. the AIA. ※ P < 0.05 vs. the SHAM.

Article Snippet: Tissue lysate (1 mg protein) was incubated with either 2 μg of mouse monoclonal anti-TLR4 antibody or 2 μg of mouse IgG isotype control antibody (5415, Cell Signaling Technology, USA) for 1 hour at room temperature.

Techniques: Expressing

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: The primary antibodies used as follows: rabbit BGN (#16409-1-AP, R&D Systems), rabbit phosphorylated Erk1/2 (pErk; #9101, Cell signaling Technology; CST, Danvers, MA, USA), rabbit total Erk1/2 (tErk; #9102, CST), rabbit phosphorylated NF-κB p65 (pNF-κB; #3033, CST), rabbit total NF-κB p65 (tNF-κB; #8242, CST), mouse TLR4 (#sc-293072, Santa Cruz Biotechmology, Dallas, TX, USA), sheep FAP (#AF3715, R&D Systems), rabbit αSMA (#ab5694, Abcam, Cambridge, UK), rabbit IL-6 (#ab6672, Abcam), mouse CD163 (#NCL-L-CD163; Leica Biosystems, Wetzlar, Germany), mouse CD206 (#sc-376108, Santa Cruz), and rabbit β-actin (#4970, CST) antibodies.

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: The primary antibodies used as follows: rabbit BGN (#16409-1-AP, R&D Systems), rabbit phosphorylated Erk1/2 (pErk; #9101, Cell signaling Technology; CST, Danvers, MA, USA), rabbit total Erk1/2 (tErk; #9102, CST), rabbit phosphorylated NF-κB p65 (pNF-κB; #3033, CST), rabbit total NF-κB p65 (tNF-κB; #8242, CST), mouse TLR4 (#sc-293072, Santa Cruz Biotechmology, Dallas, TX, USA), sheep FAP (#AF3715, R&D Systems), rabbit αSMA (#ab5694, Abcam, Cambridge, UK), rabbit IL-6 (#ab6672, Abcam), mouse CD163 (#NCL-L-CD163; Leica Biosystems, Wetzlar, Germany), mouse CD206 (#sc-376108, Santa Cruz), and rabbit β-actin (#4970, CST) antibodies.

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: The primary antibodies used as follows: rabbit BGN (#16409-1-AP, R&D Systems), rabbit phosphorylated Erk1/2 (pErk; #9101, Cell signaling Technology; CST, Danvers, MA, USA), rabbit total Erk1/2 (tErk; #9102, CST), rabbit phosphorylated NF-κB p65 (pNF-κB; #3033, CST), rabbit total NF-κB p65 (tNF-κB; #8242, CST), mouse TLR4 (#sc-293072, Santa Cruz Biotechmology, Dallas, TX, USA), sheep FAP (#AF3715, R&D Systems), rabbit αSMA (#ab5694, Abcam, Cambridge, UK), rabbit IL-6 (#ab6672, Abcam), mouse CD163 (#NCL-L-CD163; Leica Biosystems, Wetzlar, Germany), mouse CD206 (#sc-376108, Santa Cruz), and rabbit β-actin (#4970, CST) antibodies.

Techniques: Migration, Protein-Protein interactions

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