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

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

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

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

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

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

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

Journal: PLOS One

Article Title: FOXO1 transcription factor modulates airway epithelial responses to viral infection

doi: 10.1371/journal.pone.0345169

Figure Lengend Snippet: A) TLR3 mRNA expression was assessed by RT-qPCR in BEAS-2B cells transduced with FOXO1 shRNA or scrambled shRNA lentivirus. Expression levels were normalized to housekeeping gene GAPDH and expressed relative to untransduced cells (n = 12). Statistical analysis was performed with t-test ****p < 0.0001. B) TLR4 mRNA expression was analyzed by RT-qPCR in BEAS-2B cells transduced with FOXO1 shRNA and scrambled shRNA lentivirus. Expression was normalized to GAPDH and expressed relative to untransduced cells (n = 4). Statistical analysis with t-test. Representative Western blot (C) and densitometry analysis (D) of TLR3 expression for FOXO1 deficient BEAS-2B cells compared to controls, B-actin was used as a loading control (n = 6). Statistical Analysis with t-test, **p < 0.01. FOXO1 deficient cells and scrambled shRNA cells were stimulated with 50 µg/mL Poly(I:C) for 24 hours and release of IL6 (E),CCL2 (F), GM-CSF (G), IFN-λ (H), CXCL10 (I), IL8 (J), TNF-α (K), and TSLP (L) was tested with an MSD assay (n = 3). TLR3 mRNA expression was assessed by RT-qPCR in BEAS-2B (M) and NHBE (N) cells stimulated with Poly(I:C) for 8 hours in the presence or absence of the FOXO1 inhibitor AS1842856 (1 µM) (n = 5). Statistical analysis was performed using ANOVA *p < 0.05. **p < 0.01, **** P < 0.0001.

Article Snippet: Membranes were incubated with anti–FOXO1 mAb (Cell Signaling Technology, #2880) or anti-TLR3 mAb (Cell Signaling Technology, #6961) primary antibodies, followed by IRdye-conjugated goat anti-rabbit IgG (LI-COR, Lincoln, Neb); membranes were imaged with the Odyssey Infrared Imager (LI-COR).

Techniques: Expressing, Quantitative RT-PCR, Transduction, shRNA, Western Blot, Control

Journal: PLOS One

Article Title: FOXO1 transcription factor modulates airway epithelial responses to viral infection

doi: 10.1371/journal.pone.0345169

Figure Lengend Snippet: A) RT-qPCR showed increased TLR3 mRNA expression for BEAS-2B transfected with a CA-FOXO1 plasmid compared to vector control (cells transfected with an empty plasmid); GAPDH was used as a housekeeping gene (n = 6). Representative Western blot (B) and densitometry analysis (C) of TLR3 expression for BEAS-2B transfected with CA-FOXO1 plasmid compared to vector control, β-actin was used as a loading control (n = 6). Statistical Analysis with t-test, **p < 0.01. D + E) Immunofluorescence staining for BEAS-2B transduced with CA-FOXO1 shows increased FOXO1 protein in the nucleus. FOXO1 (red) was detected using an anti-FOXO1 antibody with a red-fluorescent secondary antibody, F-actin (green) with phalloidin, and nuclei (blue) with DAPI. Images were taken with an Olympus IX81 epifluorescence microscope using a 20X objective lens. Volocity Analysis was used to quantify nuclear localization of FOXO1 by measuring the mean fluorescence intensity of FOXO1 staining colocalized with DAPI. For each group 40−60 cells per slide were analyzed. Statistical Analysis was conducted with ANOVA **** p < 0.001. BEAS-2B cells transduced with FOXO1 or scrambled shRNA lentivirus were analyzed by RT-qPCR for DDX58 (RIG-I, F), MAVS (G), and MYD88 (H) mRNA expression at baseline and after Poly(I:C) stimulation (8 h and 24 h). Expression was normalized to GAPDH and expressed relative to unstimulated scrambled controls (n = 3; ANOVA). (I) NHBE cells were infected with SARS-CoV-2 in the presence or absence of a FOXO1 inhibitor. Total RNA was collected 24 h post-infection, and viral RNA levels were quantified by qRT-PCR, normalized to ACTB, and expressed relative to mock-infected cells (n = 3; paired t-test).

Article Snippet: Membranes were incubated with anti–FOXO1 mAb (Cell Signaling Technology, #2880) or anti-TLR3 mAb (Cell Signaling Technology, #6961) primary antibodies, followed by IRdye-conjugated goat anti-rabbit IgG (LI-COR, Lincoln, Neb); membranes were imaged with the Odyssey Infrared Imager (LI-COR).

Techniques: Quantitative RT-PCR, Expressing, Transfection, Plasmid Preparation, Control, Western Blot, Immunofluorescence, Staining, Transduction, Microscopy, Fluorescence, shRNA, Infection

Journal: PLOS One

Article Title: FOXO1 transcription factor modulates airway epithelial responses to viral infection

doi: 10.1371/journal.pone.0345169

Figure Lengend Snippet: A) The top panel presents FOXO1 ChIP-Seq peaks retrieved from Gene Expression Omnibus ( GSM3681486 ) in HUVEC cells and ( GSM5214707 ) in the HepG2 cell line. The bottom panel shows FOXO1 binding sites from the ChIP-Atlas visualized with IGV (Integrative Genomics Viewer). The FOXO1 motif from the HOCOMOCO database within the proximal promoter sequence of the TLR3 gene is highlighted below. B) EMSA with nuclear extracts from BEAS-2B cells incubated with FOXO1-TLR3 Promoter Oligos. Nuclear extracts from BEAS-2B cells transfected with CA-FOXO1 or plasmid control. Lane 1: dye only, Lane 2: probe only, Lane 3: nuclear extracts from CA-FOXO1 BEAS-2B cells, Lane 4: nuclear extracts from CA-FOXO1 BEAS-2B cells + cold competitor, Lane 5: nuclear extracts from vector control BEAS-2B cells, Lane 6: nuclear extracts from vector control BEAS-2B cells + cold competitor. Lane 3 shows complex I formation, which disappears in lane 4, indicating non-specific binding. C) Nuclear extracts of BEAS-2B cells transfected with CA-FOXO1 incubated with or without FOXO1 mAb shows the formation of complex I + II, but no supershift occurred. D) Incubation of protein extracts from BEAS-2B FOXO1 deficient and scrambled control lines show formation of complexes I and II but no difference is observed between cell lines.

Article Snippet: Membranes were incubated with anti–FOXO1 mAb (Cell Signaling Technology, #2880) or anti-TLR3 mAb (Cell Signaling Technology, #6961) primary antibodies, followed by IRdye-conjugated goat anti-rabbit IgG (LI-COR, Lincoln, Neb); membranes were imaged with the Odyssey Infrared Imager (LI-COR).

Techniques: ChIP-sequencing, Gene Expression, Binding Assay, Sequencing, Incubation, Transfection, Plasmid Preparation, Control

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

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