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tlr3 subcellular localization  (Santa Cruz Biotechnology)


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    Structured Review

    Santa Cruz Biotechnology tlr3 subcellular localization
    HMGB3 interacts with <t>TLR3</t> and triggers the Smad-dependent TGF-β signaling pathway via NF-kB signaling in esophageal squamous cell carcinoma (ESCC). (A) Immunohistochemistry analysis reveals TLR3 expression in 20 pairs of ESCC tissues and the corresponding paratumor tissues. (B) Western blotting analysis of EC9706 cells treated with different concentrations of poly (I:C) reveals that TLR3 positively regulates the Smad-dependent TGF-β pathway. (C) Quantitative PCR reveals the RNA expression correlation between HMGB3 and TLR3 in ESCC cell lines. (D) The co-immunoprecipitation test was conducted to explore the direct interaction between TLR3 and HMGB3. (E) The effect of HMGB3 down-regulation on NF-κB P65 nuclear expression in ECA109 was analyzed by Western blotting. (F) Quantitative PCR demonstrates the RNA levels of TGF-β and TLR3 after NF-κB P65 was up-regulated by plasmid or down-regulated by siRNA. (G) Western blotting analysis illustrates the protein levels of TGF-β and TLR3 after NF-κB P65 was up-regulated by plasmid or down-regulated by siRNA. (H, I) Chromatin immunoprecipitation and quantitative PCR assays demonstrate that TGF-β and TLR3 promoters could be directly bound by NF-κB P65. All data were expressed as mean ± standard deviation. Statistical significance is indicated as ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.
    Tlr3 Subcellular Localization, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 213 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "TGIF2-mediated HMGB3 overexpression promotes esophageal squamous cell carcinoma proliferation and metastasis through TLR3/TGF-β signaling"

    Article Title: TGIF2-mediated HMGB3 overexpression promotes esophageal squamous cell carcinoma proliferation and metastasis through TLR3/TGF-β signaling

    Journal: Genes & Diseases

    doi: 10.1016/j.gendis.2025.101987

    HMGB3 interacts with TLR3 and triggers the Smad-dependent TGF-β signaling pathway via NF-kB signaling in esophageal squamous cell carcinoma (ESCC). (A) Immunohistochemistry analysis reveals TLR3 expression in 20 pairs of ESCC tissues and the corresponding paratumor tissues. (B) Western blotting analysis of EC9706 cells treated with different concentrations of poly (I:C) reveals that TLR3 positively regulates the Smad-dependent TGF-β pathway. (C) Quantitative PCR reveals the RNA expression correlation between HMGB3 and TLR3 in ESCC cell lines. (D) The co-immunoprecipitation test was conducted to explore the direct interaction between TLR3 and HMGB3. (E) The effect of HMGB3 down-regulation on NF-κB P65 nuclear expression in ECA109 was analyzed by Western blotting. (F) Quantitative PCR demonstrates the RNA levels of TGF-β and TLR3 after NF-κB P65 was up-regulated by plasmid or down-regulated by siRNA. (G) Western blotting analysis illustrates the protein levels of TGF-β and TLR3 after NF-κB P65 was up-regulated by plasmid or down-regulated by siRNA. (H, I) Chromatin immunoprecipitation and quantitative PCR assays demonstrate that TGF-β and TLR3 promoters could be directly bound by NF-κB P65. All data were expressed as mean ± standard deviation. Statistical significance is indicated as ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.
    Figure Legend Snippet: HMGB3 interacts with TLR3 and triggers the Smad-dependent TGF-β signaling pathway via NF-kB signaling in esophageal squamous cell carcinoma (ESCC). (A) Immunohistochemistry analysis reveals TLR3 expression in 20 pairs of ESCC tissues and the corresponding paratumor tissues. (B) Western blotting analysis of EC9706 cells treated with different concentrations of poly (I:C) reveals that TLR3 positively regulates the Smad-dependent TGF-β pathway. (C) Quantitative PCR reveals the RNA expression correlation between HMGB3 and TLR3 in ESCC cell lines. (D) The co-immunoprecipitation test was conducted to explore the direct interaction between TLR3 and HMGB3. (E) The effect of HMGB3 down-regulation on NF-κB P65 nuclear expression in ECA109 was analyzed by Western blotting. (F) Quantitative PCR demonstrates the RNA levels of TGF-β and TLR3 after NF-κB P65 was up-regulated by plasmid or down-regulated by siRNA. (G) Western blotting analysis illustrates the protein levels of TGF-β and TLR3 after NF-κB P65 was up-regulated by plasmid or down-regulated by siRNA. (H, I) Chromatin immunoprecipitation and quantitative PCR assays demonstrate that TGF-β and TLR3 promoters could be directly bound by NF-κB P65. All data were expressed as mean ± standard deviation. Statistical significance is indicated as ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

    Techniques Used: Immunohistochemistry, Expressing, Western Blot, Real-time Polymerase Chain Reaction, RNA Expression, Immunoprecipitation, Plasmid Preparation, Chromatin Immunoprecipitation, Standard Deviation

    TGIF2-mediated HMGB3 overexpression promotes esophageal squamous cell carcinoma proliferation and metastasis through TLR3/TGF-β signaling.
    Figure Legend Snippet: TGIF2-mediated HMGB3 overexpression promotes esophageal squamous cell carcinoma proliferation and metastasis through TLR3/TGF-β signaling.

    Techniques Used: Over Expression

    Related Articles

    Multiple Displacement Amplification:

    Article Title: Genistein enhances TLR3-mediated apoptosis and immune signaling in breast cancer cells
    Article Snippet: Subsequently, the fixed cells were incubated with primary antibodies for 3 h, then washed twice with PBS each for 5 min, and incubated with Alexa Fluor 488-conjugated secondary antibody for 1 h. Finally, the nucleus was stained with DAPI and visualized using the EVOSTMFLoid (Life Technologies, USA) cell imaging system. .. To determine the levels of TLR3 (sc-32232, Santa Cruz), TRIF (sc-514384, Santa Cruz), IRF3 (sc-33641, Santa Cruz), p-NF-kB (3033, Cell signaling), and AP1 (MA5-15,172; Thermo Scientific) proteins in MCF-7 and MDA-MB-231 cells with the combination of GEN, Poly I:C, and GEN + Poly I:C, 1 × 10 5 cells were seeded into T75 flasks. ..

    Bioprocessing:

    Article Title: Temporally integrated transcriptome analysis reveals ASFV pathology and host response dynamics
    Article Snippet: .. The monoclonal antibodies for cGAS, TLR3, STAT1 and p-STAT1 were purchased from Santa Cruz Biotechnology, USA, and anti-β-actin, IFIH1/MDA5 and MX1 were purchased from Proteintech Biotechnology, USA. ..

    Article Title: Temporally integrated transcriptome analysis reveals ASFV pathology and host response dynamics
    Article Snippet: .. The monoclonal antibodies for cGAS, TLR3, STAT1 and p-STAT1 were purchased from Santa Cruz Biotechnology, USA, and anti-β-actin, IFIH1/MDA5 and MX1 were purchased from Proteintech Biotechnology, USA. ..

    Incubation:

    Article Title: Viral mimic polyinosine-polycytidylic acid promotes renal endothelial cell injury via HMGB1 acetylation in trichloroethylene-sensitized mice
    Article Snippet: Next, the proteins were transferred to PVDF membranes (0.45 μm, Millipore, USA) and blocked in BSA working solution for 2 hrs. .. Afterwards, antibodies against HMGB1 (Abcam, ab79823, UK, dilution 1:1000), ac-HMGB1 (ABclonal, A16002, China, dilution 1:1000), TLR3 (ZEN-BIOSCIENCE, 120101, China, dilution 1:1000), histone H3 (Santa Cruz, sc-517576, USA, dilution 1:1000), syndecan-1 (Santa Cruz, sc-390791, USA, dilution 1:1000), glypican-1 (Santa Cruz, sc-365000, USA, dilution 1:1000), or GAPDH (Abcam, ab18160, UK, dilution 1:1000) were incubated with the PVDF membrane at 4°C overnight. ..

    Article Title: Targeting Trichloroethylene-Induced Renal Endothelial Cell Injuries: A Role of Poly I:C in Amplification of HMGB1 Acetylation
    Article Snippet: Aliquots of 10 μL were subjected to separation by SDS-PAGE and subsequently transferred onto a PVDF membrane (0.45 μm pore size, Millipore, USA). .. The membranes were then treated with BSA for blocking purposes over a period of 2 h. This was followed by incubation with specific primary antibodies targeting HMGB1 (dilution 1:1500, Abcam, UK), acetylated HMGB1 (dilution 1:1500, ABclonal, China), TLR3 (dilution 1:1500, ZEN-BIOSCIENCE, China), histone H3 (dilution 1:1500, Santa Cruz, USA), syndecan-1 (dilution 1:1500, Santa Cruz, USA), glypican-1 (dilution 1:1500, Santa Cruz, USA), and GAPDH (dilution 1:1500, Abcam, UK). ..

    Membrane:

    Article Title: Viral mimic polyinosine-polycytidylic acid promotes renal endothelial cell injury via HMGB1 acetylation in trichloroethylene-sensitized mice
    Article Snippet: Next, the proteins were transferred to PVDF membranes (0.45 μm, Millipore, USA) and blocked in BSA working solution for 2 hrs. .. Afterwards, antibodies against HMGB1 (Abcam, ab79823, UK, dilution 1:1000), ac-HMGB1 (ABclonal, A16002, China, dilution 1:1000), TLR3 (ZEN-BIOSCIENCE, 120101, China, dilution 1:1000), histone H3 (Santa Cruz, sc-517576, USA, dilution 1:1000), syndecan-1 (Santa Cruz, sc-390791, USA, dilution 1:1000), glypican-1 (Santa Cruz, sc-365000, USA, dilution 1:1000), or GAPDH (Abcam, ab18160, UK, dilution 1:1000) were incubated with the PVDF membrane at 4°C overnight. ..

    Polymerase Chain Reaction:

    Article Title: Cryptosporidium uses CSpV1 to activate host type I interferon and attenuate antiparasitic defenses.
    Article Snippet: .. All sequences of PCR primers are listed in Supplementary Data 4. siRNAs, CRISPR/Cas9 and stable KO IEC4.1 cells Custom-designed RNA oligos against CSpV1-dsRNAs and scrambled siRNA-control were synthesized by Integrated DNA Technologies (Coralville, Iowa) and siRNA to Tlr3 was from Santa Cruz (sc-40259). .. Sequences of siRNAs are listed in Supplementary Data 4. siRNAs were transfected into cells with LipofectamineTM RNAiMAX Transfection Reagent according to the manufacturer’s protocol (ThermoFisher Scientific, catalogue no. 13778150).

    CRISPR:

    Article Title: Cryptosporidium uses CSpV1 to activate host type I interferon and attenuate antiparasitic defenses.
    Article Snippet: .. All sequences of PCR primers are listed in Supplementary Data 4. siRNAs, CRISPR/Cas9 and stable KO IEC4.1 cells Custom-designed RNA oligos against CSpV1-dsRNAs and scrambled siRNA-control were synthesized by Integrated DNA Technologies (Coralville, Iowa) and siRNA to Tlr3 was from Santa Cruz (sc-40259). .. Sequences of siRNAs are listed in Supplementary Data 4. siRNAs were transfected into cells with LipofectamineTM RNAiMAX Transfection Reagent according to the manufacturer’s protocol (ThermoFisher Scientific, catalogue no. 13778150).

    Synthesized:

    Article Title: Cryptosporidium uses CSpV1 to activate host type I interferon and attenuate antiparasitic defenses.
    Article Snippet: .. All sequences of PCR primers are listed in Supplementary Data 4. siRNAs, CRISPR/Cas9 and stable KO IEC4.1 cells Custom-designed RNA oligos against CSpV1-dsRNAs and scrambled siRNA-control were synthesized by Integrated DNA Technologies (Coralville, Iowa) and siRNA to Tlr3 was from Santa Cruz (sc-40259). .. Sequences of siRNAs are listed in Supplementary Data 4. siRNAs were transfected into cells with LipofectamineTM RNAiMAX Transfection Reagent according to the manufacturer’s protocol (ThermoFisher Scientific, catalogue no. 13778150).

    Blocking Assay:

    Article Title: Targeting Trichloroethylene-Induced Renal Endothelial Cell Injuries: A Role of Poly I:C in Amplification of HMGB1 Acetylation
    Article Snippet: Aliquots of 10 μL were subjected to separation by SDS-PAGE and subsequently transferred onto a PVDF membrane (0.45 μm pore size, Millipore, USA). .. The membranes were then treated with BSA for blocking purposes over a period of 2 h. This was followed by incubation with specific primary antibodies targeting HMGB1 (dilution 1:1500, Abcam, UK), acetylated HMGB1 (dilution 1:1500, ABclonal, China), TLR3 (dilution 1:1500, ZEN-BIOSCIENCE, China), histone H3 (dilution 1:1500, Santa Cruz, USA), syndecan-1 (dilution 1:1500, Santa Cruz, USA), glypican-1 (dilution 1:1500, Santa Cruz, USA), and GAPDH (dilution 1:1500, Abcam, UK). ..

    Flow Cytometry:

    Article Title: Recycled melanoma-secreted melanosomes regulate tumor-associated macrophage diversification.
    Article Snippet: The pellet was reconstituted in Red Cell Lysis Buffer (Sigma-Aldrich) according to the manufacturer’s protocol. .. The pellet were subjected to flow cytometry staining with the antibodies against APC, Alexa Fluor 488, PE, Alexa Fluor 594, and APC/Cyanine 7, specifically for the following antigens: CD45 (Miltenyi Biotech), TRAF6 (Santa Cruz), TLR3 (BioLegend), SNCA (Santa Cruz), and CD68 (BioLegend) respectively. ..

    Staining:

    Article Title: Recycled melanoma-secreted melanosomes regulate tumor-associated macrophage diversification.
    Article Snippet: The pellet was reconstituted in Red Cell Lysis Buffer (Sigma-Aldrich) according to the manufacturer’s protocol. .. The pellet were subjected to flow cytometry staining with the antibodies against APC, Alexa Fluor 488, PE, Alexa Fluor 594, and APC/Cyanine 7, specifically for the following antigens: CD45 (Miltenyi Biotech), TRAF6 (Santa Cruz), TLR3 (BioLegend), SNCA (Santa Cruz), and CD68 (BioLegend) respectively. ..



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    96
    InvivoGen tlr3 ligand
    In vivo or in vitro treatment with pDNA-LNP increased IFN-I release (A) Mice were treated as in B with PBS or 30μg of mRNA-LNP, pDNA, or pDNA-LNP. Sera were collected on day 12 and assessed for IFN-β concentration by ELISA. (B) Splenocytes from wild-type mice were incubated with media alone, a pool of Toll-like receptor agonists <t>(TLR3,</t> TLR7, and TLR9 agonists as positive control), or 1 μg per well of mRNA, mRNA-LNP, pDNA, or pDNA-LNP for 48 h. Supernatants were assessed for IFN-β by ELISA. (C) Splenocytes from wild-type mice were incubated with media alone or 1 μg per well of mRNA, mRNA-LNP, pDNA, or pDNA-LNP for 48 h. Supernatants were assessed by Luminex for CXCL10 and CCL7. (A and B) N = 3 mice per group. (C) Data are technical replicates from one mouse per group. ∗ p < 0.05 and ∗∗ p < 0.01 as assessed by one-way ANOVA with Tukey’s correction for multiple comparisons. Error bars represent mean ± SD. Experiment in (B and C) is representative of one other independent experiment.
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    (A) Bmp6 mRNA expression in primary LSECs treated with 5 ng/mL LPS or 2.5 μM heme for 6 h in the presence or absence of hepatocyte-conditioned medium. (B) Bmp6 mRNA expression in primary LSECs treated with various TLR ligands: Pam3CSK4 (TLR1/2), PGN (TLR2), Poly I:C (TLR3), LPS (TLR4), FLA-ST (TLR5), FSL1 (TLR2/6), R848 (TLR7/8), and ODN (TLR9) or vehicle control (NT, non-treated) for 6 h. (C-D) Bmp6 mRNA expression in primary LSECs treated with increasing concentrations of LPS for 6 h or with 5 ng/mL LPS for 2, 4, and 6 h. (E) Bmp6 mRNA expression in LSECs treated with 2.5 μM heme or protoporphyrin IX (PPIX) for 6 h. (F-G) Bmp6 mRNA expression in primary LSECs treated with increasing concentrations of heme for 6 h or treated with 2.5 μM heme for 2, 4, or 6 h. (H) Bmp6 mRNA expression in primary LSECs pre-treated with TAK242 (5 μM) or DMSO for 1 h, followed by 5 ng/mL LPS or 2.5 μM heme treatment for 6 h. (I) Transcription factor activity analysis by RNA-seq of LSECs treated with heme with respect to untreated controls (contrast-wise), in presence of hepatocyte-conditioned medium. (J) Bmp6 mRNA expression in primary LSECs pre-treated with CHX (5 μM) or DMSO for 1 h, followed by 5 ng/mL LPS treatment for 6 h. Cell culture experiments, except those in panel A, were always conducted in the presence of hepatocyte-conditioned medium. Gene expression levels were assessed by RT-qPCR, normalized to the housekeeping gene Rpl19 , and expressed as fold change relative to vehicle-treated controls. The dashed line (ut) represents the mRNA expression of LSECs treated with the conditions shown, in the absence of LPS or heme. Data are obtained from three or four independent experiments and displayed as mean ± SD. Statistical significance: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA or Student’s t-test. CM, conditioned medium; PPIX, protoporphyrin IX; CHX, cycloheximide.

    Journal: bioRxiv

    Article Title: Liver sinusoidal endothelial cells integrate metabolic and immune signals for MAPK-dependent BMP6 regulation and hepcidin induction

    doi: 10.64898/2026.05.07.723498

    Figure Lengend Snippet: (A) Bmp6 mRNA expression in primary LSECs treated with 5 ng/mL LPS or 2.5 μM heme for 6 h in the presence or absence of hepatocyte-conditioned medium. (B) Bmp6 mRNA expression in primary LSECs treated with various TLR ligands: Pam3CSK4 (TLR1/2), PGN (TLR2), Poly I:C (TLR3), LPS (TLR4), FLA-ST (TLR5), FSL1 (TLR2/6), R848 (TLR7/8), and ODN (TLR9) or vehicle control (NT, non-treated) for 6 h. (C-D) Bmp6 mRNA expression in primary LSECs treated with increasing concentrations of LPS for 6 h or with 5 ng/mL LPS for 2, 4, and 6 h. (E) Bmp6 mRNA expression in LSECs treated with 2.5 μM heme or protoporphyrin IX (PPIX) for 6 h. (F-G) Bmp6 mRNA expression in primary LSECs treated with increasing concentrations of heme for 6 h or treated with 2.5 μM heme for 2, 4, or 6 h. (H) Bmp6 mRNA expression in primary LSECs pre-treated with TAK242 (5 μM) or DMSO for 1 h, followed by 5 ng/mL LPS or 2.5 μM heme treatment for 6 h. (I) Transcription factor activity analysis by RNA-seq of LSECs treated with heme with respect to untreated controls (contrast-wise), in presence of hepatocyte-conditioned medium. (J) Bmp6 mRNA expression in primary LSECs pre-treated with CHX (5 μM) or DMSO for 1 h, followed by 5 ng/mL LPS treatment for 6 h. Cell culture experiments, except those in panel A, were always conducted in the presence of hepatocyte-conditioned medium. Gene expression levels were assessed by RT-qPCR, normalized to the housekeeping gene Rpl19 , and expressed as fold change relative to vehicle-treated controls. The dashed line (ut) represents the mRNA expression of LSECs treated with the conditions shown, in the absence of LPS or heme. Data are obtained from three or four independent experiments and displayed as mean ± SD. Statistical significance: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA or Student’s t-test. CM, conditioned medium; PPIX, protoporphyrin IX; CHX, cycloheximide.

    Article Snippet: The TLR ligands Pam3CSK4 (TLR2:1) (#tlrl-pms), PGN-SA (TLR2) (#tlrl-pgns2), Poly I:C (TLR3) (#tlrl-picw), FLA-ST (TLR5) (#tlrl-stfla), FSL1 (TLR2:TLR6) (tlrl-fsl), R848 (TLR7:8) (#tlrl-r848-1), ODN (TLR9) (#tlrl-1826) and the MAPK inhibitor SP600125 (#tlrl-sp60) were purchased form Invivogen and diluted in PBS (TLR ligands) or DMSO (SP600125).

    Techniques: Expressing, Control, Activity Assay, RNA Sequencing, Cell Culture, Gene Expression, Quantitative RT-PCR

    HMGB3 interacts with TLR3 and triggers the Smad-dependent TGF-β signaling pathway via NF-kB signaling in esophageal squamous cell carcinoma (ESCC). (A) Immunohistochemistry analysis reveals TLR3 expression in 20 pairs of ESCC tissues and the corresponding paratumor tissues. (B) Western blotting analysis of EC9706 cells treated with different concentrations of poly (I:C) reveals that TLR3 positively regulates the Smad-dependent TGF-β pathway. (C) Quantitative PCR reveals the RNA expression correlation between HMGB3 and TLR3 in ESCC cell lines. (D) The co-immunoprecipitation test was conducted to explore the direct interaction between TLR3 and HMGB3. (E) The effect of HMGB3 down-regulation on NF-κB P65 nuclear expression in ECA109 was analyzed by Western blotting. (F) Quantitative PCR demonstrates the RNA levels of TGF-β and TLR3 after NF-κB P65 was up-regulated by plasmid or down-regulated by siRNA. (G) Western blotting analysis illustrates the protein levels of TGF-β and TLR3 after NF-κB P65 was up-regulated by plasmid or down-regulated by siRNA. (H, I) Chromatin immunoprecipitation and quantitative PCR assays demonstrate that TGF-β and TLR3 promoters could be directly bound by NF-κB P65. All data were expressed as mean ± standard deviation. Statistical significance is indicated as ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

    Journal: Genes & Diseases

    Article Title: TGIF2-mediated HMGB3 overexpression promotes esophageal squamous cell carcinoma proliferation and metastasis through TLR3/TGF-β signaling

    doi: 10.1016/j.gendis.2025.101987

    Figure Lengend Snippet: HMGB3 interacts with TLR3 and triggers the Smad-dependent TGF-β signaling pathway via NF-kB signaling in esophageal squamous cell carcinoma (ESCC). (A) Immunohistochemistry analysis reveals TLR3 expression in 20 pairs of ESCC tissues and the corresponding paratumor tissues. (B) Western blotting analysis of EC9706 cells treated with different concentrations of poly (I:C) reveals that TLR3 positively regulates the Smad-dependent TGF-β pathway. (C) Quantitative PCR reveals the RNA expression correlation between HMGB3 and TLR3 in ESCC cell lines. (D) The co-immunoprecipitation test was conducted to explore the direct interaction between TLR3 and HMGB3. (E) The effect of HMGB3 down-regulation on NF-κB P65 nuclear expression in ECA109 was analyzed by Western blotting. (F) Quantitative PCR demonstrates the RNA levels of TGF-β and TLR3 after NF-κB P65 was up-regulated by plasmid or down-regulated by siRNA. (G) Western blotting analysis illustrates the protein levels of TGF-β and TLR3 after NF-κB P65 was up-regulated by plasmid or down-regulated by siRNA. (H, I) Chromatin immunoprecipitation and quantitative PCR assays demonstrate that TGF-β and TLR3 promoters could be directly bound by NF-κB P65. All data were expressed as mean ± standard deviation. Statistical significance is indicated as ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

    Article Snippet: To assess TLR3 subcellular localization, ECA109 cells were stained with an anti-TLR3 antibody (Santa Cruz, Cat. sc-32232; dilution 1:20) at 4 °C for 60 min under either permeabilized or non-permeabilized conditions.

    Techniques: Immunohistochemistry, Expressing, Western Blot, Real-time Polymerase Chain Reaction, RNA Expression, Immunoprecipitation, Plasmid Preparation, Chromatin Immunoprecipitation, Standard Deviation

    TGIF2-mediated HMGB3 overexpression promotes esophageal squamous cell carcinoma proliferation and metastasis through TLR3/TGF-β signaling.

    Journal: Genes & Diseases

    Article Title: TGIF2-mediated HMGB3 overexpression promotes esophageal squamous cell carcinoma proliferation and metastasis through TLR3/TGF-β signaling

    doi: 10.1016/j.gendis.2025.101987

    Figure Lengend Snippet: TGIF2-mediated HMGB3 overexpression promotes esophageal squamous cell carcinoma proliferation and metastasis through TLR3/TGF-β signaling.

    Article Snippet: To assess TLR3 subcellular localization, ECA109 cells were stained with an anti-TLR3 antibody (Santa Cruz, Cat. sc-32232; dilution 1:20) at 4 °C for 60 min under either permeabilized or non-permeabilized conditions.

    Techniques: Over Expression

    HMGB3 interacts with TLR3 and triggers the Smad-dependent TGF-β signaling pathway via NF-kB signaling in esophageal squamous cell carcinoma (ESCC). (A) Immunohistochemistry analysis reveals TLR3 expression in 20 pairs of ESCC tissues and the corresponding paratumor tissues. (B) Western blotting analysis of EC9706 cells treated with different concentrations of poly (I:C) reveals that TLR3 positively regulates the Smad-dependent TGF-β pathway. (C) Quantitative PCR reveals the RNA expression correlation between HMGB3 and TLR3 in ESCC cell lines. (D) The co-immunoprecipitation test was conducted to explore the direct interaction between TLR3 and HMGB3. (E) The effect of HMGB3 down-regulation on NF-κB P65 nuclear expression in ECA109 was analyzed by Western blotting. (F) Quantitative PCR demonstrates the RNA levels of TGF-β and TLR3 after NF-κB P65 was up-regulated by plasmid or down-regulated by siRNA. (G) Western blotting analysis illustrates the protein levels of TGF-β and TLR3 after NF-κB P65 was up-regulated by plasmid or down-regulated by siRNA. (H, I) Chromatin immunoprecipitation and quantitative PCR assays demonstrate that TGF-β and TLR3 promoters could be directly bound by NF-κB P65. All data were expressed as mean ± standard deviation. Statistical significance is indicated as ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

    Journal: Genes & Diseases

    Article Title: TGIF2-mediated HMGB3 overexpression promotes esophageal squamous cell carcinoma proliferation and metastasis through TLR3/TGF-β signaling

    doi: 10.1016/j.gendis.2025.101987

    Figure Lengend Snippet: HMGB3 interacts with TLR3 and triggers the Smad-dependent TGF-β signaling pathway via NF-kB signaling in esophageal squamous cell carcinoma (ESCC). (A) Immunohistochemistry analysis reveals TLR3 expression in 20 pairs of ESCC tissues and the corresponding paratumor tissues. (B) Western blotting analysis of EC9706 cells treated with different concentrations of poly (I:C) reveals that TLR3 positively regulates the Smad-dependent TGF-β pathway. (C) Quantitative PCR reveals the RNA expression correlation between HMGB3 and TLR3 in ESCC cell lines. (D) The co-immunoprecipitation test was conducted to explore the direct interaction between TLR3 and HMGB3. (E) The effect of HMGB3 down-regulation on NF-κB P65 nuclear expression in ECA109 was analyzed by Western blotting. (F) Quantitative PCR demonstrates the RNA levels of TGF-β and TLR3 after NF-κB P65 was up-regulated by plasmid or down-regulated by siRNA. (G) Western blotting analysis illustrates the protein levels of TGF-β and TLR3 after NF-κB P65 was up-regulated by plasmid or down-regulated by siRNA. (H, I) Chromatin immunoprecipitation and quantitative PCR assays demonstrate that TGF-β and TLR3 promoters could be directly bound by NF-κB P65. All data were expressed as mean ± standard deviation. Statistical significance is indicated as ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

    Article Snippet: To assess TLR3 subcellular localization, ECA109 cells were stained with an anti-TLR3 antibody (Santa Cruz, Cat. sc-32232; dilution 1:20) at 4 °C for 60 min under either permeabilized or non-permeabilized conditions.

    Techniques: Immunohistochemistry, Expressing, Western Blot, Real-time Polymerase Chain Reaction, RNA Expression, Immunoprecipitation, Plasmid Preparation, Chromatin Immunoprecipitation, Standard Deviation

    TGIF2-mediated HMGB3 overexpression promotes esophageal squamous cell carcinoma proliferation and metastasis through TLR3/TGF-β signaling.

    Journal: Genes & Diseases

    Article Title: TGIF2-mediated HMGB3 overexpression promotes esophageal squamous cell carcinoma proliferation and metastasis through TLR3/TGF-β signaling

    doi: 10.1016/j.gendis.2025.101987

    Figure Lengend Snippet: TGIF2-mediated HMGB3 overexpression promotes esophageal squamous cell carcinoma proliferation and metastasis through TLR3/TGF-β signaling.

    Article Snippet: To assess TLR3 subcellular localization, ECA109 cells were stained with an anti-TLR3 antibody (Santa Cruz, Cat. sc-32232; dilution 1:20) at 4 °C for 60 min under either permeabilized or non-permeabilized conditions.

    Techniques: Over Expression

    HMGB3 and TGIF2 activate TGF-β signaling in esophageal squamous cell carcinoma (ESCC). (A) RNA sequencing analysis was performed on ECA109 cells infected with LV-control and LV-shHMGB3-1. The RNA sequencing volcano plot highlights 168 genes as up-regulated and 141 genes as down-regulated. Blue dots represent genes with lower expression in ECA109-control compared with ECA109-shHMGB3-1, whereas red dots indicate higher expressed genes. (B) The heatmap generated from RNA sequencing data displays 168 up-regulated genes and 141 down-regulated genes when comparing LV-control with LV-shHMGB3-1 in ECA109 cells. Red highlights indicate up-regulated genes, whereas blue highlights indicate down-regulated genes. (C) KEGG analysis was used to identify the top 30 most relevant pathways. (D) Western blotting analysis demonstrates that the HMGB3 positively regulates Smad-dependent TGF-β signaling. (E) The proliferative capacity of ESCC cells after modifying the expression of TGF-β was demonstrated by the CCK-8 assay, with optical density (OD) measurements obtained daily for 5 days. (F) The migratory and invasive abilities of ESCC cells after modifying the expression of TGF-β were demonstrated by the Transwell analysis. The left panel illustrates the results, whereas the right panel shows the number of migrated and invasive cells calculated and compared, respectively. (G) Western blotting analysis demonstrates that TGIF2 positively regulates Smad-dependent TGF-β signaling. (H) WB analysis demonstrates that TGIF2 positively regulates the Smad-dependent TGF-β pathway in an HMGB3-dependent manner. All data were expressed as mean ± standard deviation. Statistical significance is indicated as ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, # P < 0.05, ## P < 0.01, and ### P < 0.001.

    Journal: Genes & Diseases

    Article Title: TGIF2-mediated HMGB3 overexpression promotes esophageal squamous cell carcinoma proliferation and metastasis through TLR3/TGF-β signaling

    doi: 10.1016/j.gendis.2025.101987

    Figure Lengend Snippet: HMGB3 and TGIF2 activate TGF-β signaling in esophageal squamous cell carcinoma (ESCC). (A) RNA sequencing analysis was performed on ECA109 cells infected with LV-control and LV-shHMGB3-1. The RNA sequencing volcano plot highlights 168 genes as up-regulated and 141 genes as down-regulated. Blue dots represent genes with lower expression in ECA109-control compared with ECA109-shHMGB3-1, whereas red dots indicate higher expressed genes. (B) The heatmap generated from RNA sequencing data displays 168 up-regulated genes and 141 down-regulated genes when comparing LV-control with LV-shHMGB3-1 in ECA109 cells. Red highlights indicate up-regulated genes, whereas blue highlights indicate down-regulated genes. (C) KEGG analysis was used to identify the top 30 most relevant pathways. (D) Western blotting analysis demonstrates that the HMGB3 positively regulates Smad-dependent TGF-β signaling. (E) The proliferative capacity of ESCC cells after modifying the expression of TGF-β was demonstrated by the CCK-8 assay, with optical density (OD) measurements obtained daily for 5 days. (F) The migratory and invasive abilities of ESCC cells after modifying the expression of TGF-β were demonstrated by the Transwell analysis. The left panel illustrates the results, whereas the right panel shows the number of migrated and invasive cells calculated and compared, respectively. (G) Western blotting analysis demonstrates that TGIF2 positively regulates Smad-dependent TGF-β signaling. (H) WB analysis demonstrates that TGIF2 positively regulates the Smad-dependent TGF-β pathway in an HMGB3-dependent manner. All data were expressed as mean ± standard deviation. Statistical significance is indicated as ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, # P < 0.05, ## P < 0.01, and ### P < 0.001.

    Article Snippet: To assess TLR3 subcellular localization, ECA109 cells were stained with an anti-TLR3 antibody (Santa Cruz, Cat. sc-32232; dilution 1:20) at 4 °C for 60 min under either permeabilized or non-permeabilized conditions.

    Techniques: RNA Sequencing, Infection, Control, Expressing, Generated, Western Blot, CCK-8 Assay, Standard Deviation

    HMGB3 interacts with TLR3 and triggers the Smad-dependent TGF-β signaling pathway via NF-kB signaling in esophageal squamous cell carcinoma (ESCC). (A) Immunohistochemistry analysis reveals TLR3 expression in 20 pairs of ESCC tissues and the corresponding paratumor tissues. (B) Western blotting analysis of EC9706 cells treated with different concentrations of poly (I:C) reveals that TLR3 positively regulates the Smad-dependent TGF-β pathway. (C) Quantitative PCR reveals the RNA expression correlation between HMGB3 and TLR3 in ESCC cell lines. (D) The co-immunoprecipitation test was conducted to explore the direct interaction between TLR3 and HMGB3. (E) The effect of HMGB3 down-regulation on NF-κB P65 nuclear expression in ECA109 was analyzed by Western blotting. (F) Quantitative PCR demonstrates the RNA levels of TGF-β and TLR3 after NF-κB P65 was up-regulated by plasmid or down-regulated by siRNA. (G) Western blotting analysis illustrates the protein levels of TGF-β and TLR3 after NF-κB P65 was up-regulated by plasmid or down-regulated by siRNA. (H, I) Chromatin immunoprecipitation and quantitative PCR assays demonstrate that TGF-β and TLR3 promoters could be directly bound by NF-κB P65. All data were expressed as mean ± standard deviation. Statistical significance is indicated as ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

    Journal: Genes & Diseases

    Article Title: TGIF2-mediated HMGB3 overexpression promotes esophageal squamous cell carcinoma proliferation and metastasis through TLR3/TGF-β signaling

    doi: 10.1016/j.gendis.2025.101987

    Figure Lengend Snippet: HMGB3 interacts with TLR3 and triggers the Smad-dependent TGF-β signaling pathway via NF-kB signaling in esophageal squamous cell carcinoma (ESCC). (A) Immunohistochemistry analysis reveals TLR3 expression in 20 pairs of ESCC tissues and the corresponding paratumor tissues. (B) Western blotting analysis of EC9706 cells treated with different concentrations of poly (I:C) reveals that TLR3 positively regulates the Smad-dependent TGF-β pathway. (C) Quantitative PCR reveals the RNA expression correlation between HMGB3 and TLR3 in ESCC cell lines. (D) The co-immunoprecipitation test was conducted to explore the direct interaction between TLR3 and HMGB3. (E) The effect of HMGB3 down-regulation on NF-κB P65 nuclear expression in ECA109 was analyzed by Western blotting. (F) Quantitative PCR demonstrates the RNA levels of TGF-β and TLR3 after NF-κB P65 was up-regulated by plasmid or down-regulated by siRNA. (G) Western blotting analysis illustrates the protein levels of TGF-β and TLR3 after NF-κB P65 was up-regulated by plasmid or down-regulated by siRNA. (H, I) Chromatin immunoprecipitation and quantitative PCR assays demonstrate that TGF-β and TLR3 promoters could be directly bound by NF-κB P65. All data were expressed as mean ± standard deviation. Statistical significance is indicated as ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

    Article Snippet: To assess TLR3 subcellular localization, ECA109 cells were stained with an anti-TLR3 antibody (Santa Cruz, Cat. sc-32232; dilution 1:20) at 4 °C for 60 min under either permeabilized or non-permeabilized conditions.

    Techniques: Immunohistochemistry, Expressing, Western Blot, Real-time Polymerase Chain Reaction, RNA Expression, Immunoprecipitation, Plasmid Preparation, Chromatin Immunoprecipitation, Standard Deviation

    In vivo or in vitro treatment with pDNA-LNP increased IFN-I release (A) Mice were treated as in B with PBS or 30μg of mRNA-LNP, pDNA, or pDNA-LNP. Sera were collected on day 12 and assessed for IFN-β concentration by ELISA. (B) Splenocytes from wild-type mice were incubated with media alone, a pool of Toll-like receptor agonists (TLR3, TLR7, and TLR9 agonists as positive control), or 1 μg per well of mRNA, mRNA-LNP, pDNA, or pDNA-LNP for 48 h. Supernatants were assessed for IFN-β by ELISA. (C) Splenocytes from wild-type mice were incubated with media alone or 1 μg per well of mRNA, mRNA-LNP, pDNA, or pDNA-LNP for 48 h. Supernatants were assessed by Luminex for CXCL10 and CCL7. (A and B) N = 3 mice per group. (C) Data are technical replicates from one mouse per group. ∗ p < 0.05 and ∗∗ p < 0.01 as assessed by one-way ANOVA with Tukey’s correction for multiple comparisons. Error bars represent mean ± SD. Experiment in (B and C) is representative of one other independent experiment.

    Journal: Molecular Therapy Advances

    Article Title: Plasmid DNA vaccines encapsulated in lipid nanoparticles elicit STING-dependent type 1 interferon release

    doi: 10.1016/j.omta.2026.201698

    Figure Lengend Snippet: In vivo or in vitro treatment with pDNA-LNP increased IFN-I release (A) Mice were treated as in B with PBS or 30μg of mRNA-LNP, pDNA, or pDNA-LNP. Sera were collected on day 12 and assessed for IFN-β concentration by ELISA. (B) Splenocytes from wild-type mice were incubated with media alone, a pool of Toll-like receptor agonists (TLR3, TLR7, and TLR9 agonists as positive control), or 1 μg per well of mRNA, mRNA-LNP, pDNA, or pDNA-LNP for 48 h. Supernatants were assessed for IFN-β by ELISA. (C) Splenocytes from wild-type mice were incubated with media alone or 1 μg per well of mRNA, mRNA-LNP, pDNA, or pDNA-LNP for 48 h. Supernatants were assessed by Luminex for CXCL10 and CCL7. (A and B) N = 3 mice per group. (C) Data are technical replicates from one mouse per group. ∗ p < 0.05 and ∗∗ p < 0.01 as assessed by one-way ANOVA with Tukey’s correction for multiple comparisons. Error bars represent mean ± SD. Experiment in (B and C) is representative of one other independent experiment.

    Article Snippet: In some cases, cells were also incubated with 10 μg/mL TLR3 ligand (poly I:C, InVivoGen #vac-pic, San Diego, CA), 3 μg/mL TLR7 ligand (Gardiquimod, InVivoGen #tlrl-gdqs-1), 5 μM TLR9 ligand (ODN1826, Integrated DNA Technologies, San Diego, CA), 0.5 μM STING agonist (diABZI, InVivoGen #tlrl-diabzi-2; a generous gift from Dr.

    Techniques: In Vivo, In Vitro, Concentration Assay, Enzyme-linked Immunosorbent Assay, Incubation, Positive Control, Luminex

    Increased IFN-β levels were not due to TLR3, 7, or 9 signaling but were at least partially mediated by STING signaling (A) Splenocytes from wild-type, TLR3 knockout (TLR3KO), TLR7KO, or TLR9KO were incubated with indicated TLR ligands or 1 μg per well of mRNA-LNP, mRNA, pDNA, or pDNA-LNP for 48 h. Supernatants were assessed for IFN-β by ELISA. N = 3 mice per strain. (B) Splenocytes from wild-type or STING knockout mice were incubated with a pool of TLR ligands, diABZI (STING agonist), or 1 μg per well of mRNA, mRNA-LNP, pDNA, or pDNA-LNP for 48 h. Supernatants were assessed for IFN-β by ELISA. N = 3 mice/strain. Supernatants were assessed for IFN-β (by ELISA), or CXCL10, CCL7, or CCL5 (by Luminex). N = 3 mice per strain (ELISA) or N = 1 mouse per strain assessed in technical replicates (Luminex studies). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 as assessed by one-way ANOVA with Tukey’s correction for multiple comparison. Results are from one experiment (A, and Luminex studies in B) or are representative of two independent experiments (IFN-β ELISA).

    Journal: Molecular Therapy Advances

    Article Title: Plasmid DNA vaccines encapsulated in lipid nanoparticles elicit STING-dependent type 1 interferon release

    doi: 10.1016/j.omta.2026.201698

    Figure Lengend Snippet: Increased IFN-β levels were not due to TLR3, 7, or 9 signaling but were at least partially mediated by STING signaling (A) Splenocytes from wild-type, TLR3 knockout (TLR3KO), TLR7KO, or TLR9KO were incubated with indicated TLR ligands or 1 μg per well of mRNA-LNP, mRNA, pDNA, or pDNA-LNP for 48 h. Supernatants were assessed for IFN-β by ELISA. N = 3 mice per strain. (B) Splenocytes from wild-type or STING knockout mice were incubated with a pool of TLR ligands, diABZI (STING agonist), or 1 μg per well of mRNA, mRNA-LNP, pDNA, or pDNA-LNP for 48 h. Supernatants were assessed for IFN-β by ELISA. N = 3 mice/strain. Supernatants were assessed for IFN-β (by ELISA), or CXCL10, CCL7, or CCL5 (by Luminex). N = 3 mice per strain (ELISA) or N = 1 mouse per strain assessed in technical replicates (Luminex studies). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 as assessed by one-way ANOVA with Tukey’s correction for multiple comparison. Results are from one experiment (A, and Luminex studies in B) or are representative of two independent experiments (IFN-β ELISA).

    Article Snippet: In some cases, cells were also incubated with 10 μg/mL TLR3 ligand (poly I:C, InVivoGen #vac-pic, San Diego, CA), 3 μg/mL TLR7 ligand (Gardiquimod, InVivoGen #tlrl-gdqs-1), 5 μM TLR9 ligand (ODN1826, Integrated DNA Technologies, San Diego, CA), 0.5 μM STING agonist (diABZI, InVivoGen #tlrl-diabzi-2; a generous gift from Dr.

    Techniques: Knock-Out, Incubation, Enzyme-linked Immunosorbent Assay, Luminex, Comparison