tlr4 inhibitor  (MedChemExpress)

Journal: Molecular Therapy. Nucleic Acids

Article Title: Hemoglobin inhibits fibroblast-to-cardiomyocyte reprogramming via TLR2/TLR4-dependent chromatin compaction

doi: 10.1016/j.omtn.2026.102900

Figure Lengend Snippet: Hemoglobin activates TLR signaling in cardiac fibroblasts (A) Cardiac fibroblasts were incubated with Hb (5 mg/mL) for the indicated times. Protein extracts were analyzed by immunoblotting for p-NFκB and p-MAPK. Gapdh was used as a loading control. Representative immunoblots are shown on the left-hand side. Quantification was performed by normalizing p-NFκB and p-MAPK band densities with those of the loading control. N = 4. One-sample t tests were used to compare groups to the control group (∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05; ns, not significant). (B) Cardiac fibroblasts were incubated either vehicle or with varying concentrations of a TLR4 pharmacological inhibitor for 3 h. Hb (5 mg/mL) was then added. After 1 h, protein extracts were isolated and subsequently analyzed by immunoblotting for p-NFκB. Gapdh was used as a loading control. Representative immunoblots are shown on the left-hand side. Quantification was performed by normalizing p-NFκB band densities with those of the loading control. N = 4. One-sample t tests were used to compare groups to the control group (∗ p < 0.05; ns, not significant). (C) Cardiac fibroblasts were incubated with either LPS or Hb for 24 h, after which expression of the indicated pro-inflammatory cytokines was determined by qPCR. Expression values are shown relative to the housekeeping gene Gapdh. N = 6. ANOVA with Tukey post-hoc tests were used to determine significance (∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05; ns, not significant).

Article Snippet: Hb (Millipore Sigma, H2625), TLR2 signaling inhibitor-TL2-C29 (InvivoGen, catalog no. inh-c29), and TLR4 inhibitor-CLI-095 (InvivoGen, catalog no. tlrl-cli95-4) were used.

Techniques: Incubation, Western Blot, Control, Isolation, Expressing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Hemoglobin inhibits fibroblast-to-cardiomyocyte reprogramming via TLR2/TLR4-dependent chromatin compaction

doi: 10.1016/j.omtn.2026.102900

Figure Lengend Snippet: Hemoglobin mediates gene repression through TLR2 and TLR4 (A and B) A study was conducted to determine the effect of hemoglobin (Hb) on (A) fibroblast-to-cardiomyocyte reprogramming and (B) fibroblast gene expression. With respect to fibroblast-to-cardiomyocyte reprogramming, cardiac fibroblasts were transfected with either miR combo or a non-targeting control miR. 24 h later, the cells were incubated with vehicle, a TLR2 pharmacological inhibitor, a TLR4 pharmacological inhibitor, or a combination of both pharmacological inhibitors for 3 h. After incubation with the indicated pharmacological inhibitors, Hb was added (5 mg/mL) to the media. All media was replaced the next day. Fourteen days after miR transfection, cells were analyzed for expression of the indicated cardiomyocyte specific genes by qPCR. Expression values were normalized to the housekeeping gene Gapdh. N = 6–10. One-sample t tests were used to compare groups to the control group (∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05; ns, not significant). t test was used to determine the significance between the miR combo groups (## p < 0.01, # p < 0.05; ns, not significant). With respect to fibroblast gene expression, cardiac fibroblasts were incubated with vehicle, a TLR2 pharmacological inhibitor, a TLR4 pharmacological inhibitor, or a combination of both pharmacological inhibitors for 3 h. After incubation with the indicated pharmacological inhibitors, Hb was added (5 mg/mL) to the media. All media was replaced the next day. Expression of the indicated fibroblast-specific genes was determined by qPCR and normalized to the housekeeping gene Gapdh. N = 10. One-sample t tests were used to compare groups to the control group (∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05; ns, not significant).

Article Snippet: Hb (Millipore Sigma, H2625), TLR2 signaling inhibitor-TL2-C29 (InvivoGen, catalog no. inh-c29), and TLR4 inhibitor-CLI-095 (InvivoGen, catalog no. tlrl-cli95-4) were used.

Techniques: Gene Expression, Transfection, Control, Incubation, Expressing

Journal: Hepatology Communications

Article Title: CRISPLD2 protects against liver inflammation and fibrosis via GRP78 to repress HMGB1/TLR4 axis–mediated STING palmitoylation

doi: 10.1097/HC9.0000000000000954

Figure Lengend Snippet: Activation of the HMGB1/TLR4 axis induced hepatocyte inflammatory response and fibrosis. (A) Primary hepatocytes were treated with 5 μg/mL HMGB1 for 3, 9, 18, 24, and 48 hours. IL-6, IL-1β, and TNF-α levels in the culture medium were assessed by ELISA. (B) JS-1 cells were co-cultured with primary hepatocytes treated with 5 μg/mL HMGB1 for 3, 9, 18, 24, and 48 hours, and α-SMA, FN, and col1a1 protein abundance in JS-1 cells was determined by western blotting. (C) Primary hepatocytes were added with 5 μg/mL HMGB1 together with or without 10 nM TAK-242 for 24 hours, and IL-6, IL-1β, and TNF-α levels in the culture medium were detected by ELISA. (D) Primary hepatocytes with various treatments were co-cultured with JS-1 cells, and western blotting analysis of α-SMA, FN, and col1a1 protein levels in JS-1 cells. n=3. One-way ANOVA was performed for statistical analysis. * p <0.05, ** p <0.01, and *** p <0.001. Abbreviations: α-SMA, alpha-smooth muscle actin; ANOVA, analysis of variance; COL1A1, collagen type I alpha 1 chain; ELISA, enzyme-linked immunosorbent assay; FN, fibronectin; HMGB1, high mobility group box 1; IL, interleukin; TNF-α, tumor necrosis factor-alpha.

Article Snippet: The primary hepatocytes were treated with 10 nM TLR4 inhibitor (TAK-242, HY-11109; MCE, NJ, USA) for 24 hours; 100 μM palmitoylation inhibitor, 2-bromopalmitate (2-BP, HY-111770; MCE) for 2 and 4 hours; 10 μM palmitoylation enhancer, palmostatin B (HY-120911; MCE) for 2, 4, and 8 hours; 20 μM proteasome inhibitor MG132 (HY-13259; MCE); and 10 mM autophagy–lysosome inhibitor chloroquine (CQ, HY-17589A; MCE) for 4 hours.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Quantitative Proteomics, Western Blot

Journal: Hepatology Communications

Article Title: CRISPLD2 protects against liver inflammation and fibrosis via GRP78 to repress HMGB1/TLR4 axis–mediated STING palmitoylation

doi: 10.1097/HC9.0000000000000954

Figure Lengend Snippet: STING palmitoylation at the C64 site was involved in HMGB1/TLR4 axis–mediated hepatocyte inflammatory response and fibrosis. (A) Primary hepatocytes were treated with 5 μg/mL HMGB1 combined with or without 10 nM TAK-242 for 24 hours, and the ABE assay evaluated STING palmitoylation level. The cells were added with hydroxylamine (+HAM) buffer or lysis buffer (−HAM). Palm-STING, STING palmitoylation. (B) Primary hepatocytes were treated with 100 μM 2-BP (a palmitoylation inhibitor) for 2–4 hours, and ABE assay analysis of STING palmitoylation level. The cells were added with hydroxylamine (+HAM) buffer or lysis buffer (−HAM). Palm-STING, STING palmitoylation. (C) STING palmitoylation level in primary hepatocytes exposed to 10 μM palmostatin B (a palmitoylation enhancer) for 2, 4, and 8 hours was analyzed by ABE assay. The cells were added with hydroxylamine (+HAM) buffer or lysis buffer (−HAM). Palm-STING, STING palmitoylation. (D) Conservative analysis of STING protein sequences among different species. (E) HEK293T cells were transfected with STING-WT or STING-C64A, and the Acyl-RAC assay determined STING palmitoylation level. Primary hepatocytes infected with lentiviruses carrying STING-WT or STING-C64A and treated with 5 μg/mL HMGB1, followed by co-culture with JS-1 cells. (F) ELISA measured IL-6, IL-1β, and TNF-α levels in the supernatant of primary hepatocytes. (G) Western blotting analysis of α-SMA, FN, and col1a1 protein abundance in JS-1 cells. n=3. One-way ANOVA was performed for statistical analysis. * p <0.05, ** p <0.01, and *** p <0.001. Abbreviations: ABE, Acyl-Biotin Exchange; α-SMA, alpha-smooth muscle actin; ANOVA, analysis of variance; COL1A1, collagen type I alpha 1 chain; ELISA, enzyme-linked immunosorbent assay; FN, fibronectin; HAM, hydroxylamine; HMGB1, high mobility group box 1; 2-BP, 2-bromopalmitate; STING, stimulator of interferon genes; STING-WT, wild-type STING.

Article Snippet: The primary hepatocytes were treated with 10 nM TLR4 inhibitor (TAK-242, HY-11109; MCE, NJ, USA) for 24 hours; 100 μM palmitoylation inhibitor, 2-bromopalmitate (2-BP, HY-111770; MCE) for 2 and 4 hours; 10 μM palmitoylation enhancer, palmostatin B (HY-120911; MCE) for 2, 4, and 8 hours; 20 μM proteasome inhibitor MG132 (HY-13259; MCE); and 10 mM autophagy–lysosome inhibitor chloroquine (CQ, HY-17589A; MCE) for 4 hours.

Techniques: Lysis, Transfection, Infection, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Quantitative Proteomics

Journal: Hepatology Communications

Article Title: CRISPLD2 protects against liver inflammation and fibrosis via GRP78 to repress HMGB1/TLR4 axis–mediated STING palmitoylation

doi: 10.1097/HC9.0000000000000954

Figure Lengend Snippet: CRISPLD2 inactivated the HMGB1/TLR4 axis to repress STING palmitoylation, thus suppressing hepatocyte inflammatory response and fibrosis. HMGB1-treated primary hepatocytes were further stimulated with 1, 5, 10, and 20 μg/mL CRISPLD2 for 24 hours, followed by co-culture with JS-1 cells. (A) ELISA determined IL-6, IL-1β, and TNF-α levels in the supernatant of primary hepatocytes. (B) α-SMA, FN, and col1a1 protein abundance in JS-1 cells was assessed by western blotting. (C) STING palmitoylation levels were evaluated by the ABE assay. The cells were added with hydroxylamine (+HAM) buffer or lysis buffer (−HAM). Palm-STING, STING palmitoylation. n=3. One-way ANOVA was performed for statistical analysis. * p <0.05, ** p <0.01, and *** p <0.001. ABE, Acyl-Biotin Exchange; α-SMA, alpha-smooth muscle actin; ANOVA, analysis of variance; COL1A1, collagen type I alpha 1 chain; CRISPLD2, cysteine-rich secreted protein LCCL domain 2; ELISA, enzyme-linked immunosorbent assay; FN, fibronectin; HAM, hydroxylamine; HMGB1, high mobility group box 1; STING, stimulator of interferon genes.

Article Snippet: The primary hepatocytes were treated with 10 nM TLR4 inhibitor (TAK-242, HY-11109; MCE, NJ, USA) for 24 hours; 100 μM palmitoylation inhibitor, 2-bromopalmitate (2-BP, HY-111770; MCE) for 2 and 4 hours; 10 μM palmitoylation enhancer, palmostatin B (HY-120911; MCE) for 2, 4, and 8 hours; 20 μM proteasome inhibitor MG132 (HY-13259; MCE); and 10 mM autophagy–lysosome inhibitor chloroquine (CQ, HY-17589A; MCE) for 4 hours.

Techniques: Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Quantitative Proteomics, Western Blot, Lysis

Journal: Hepatology Communications

Article Title: CRISPLD2 protects against liver inflammation and fibrosis via GRP78 to repress HMGB1/TLR4 axis–mediated STING palmitoylation

doi: 10.1097/HC9.0000000000000954

Figure Lengend Snippet: CRISPLD2 degraded TLR4 via an autophagic–lysosomal pathway. (A) Primary hepatocytes were treated with 5 μg/mL HMGB1, 10 μg/mL CRISPLD2, or a combination of them. Western blotting analysis of TLR4 protein level. (B) Primary hepatocytes were treated with 10 μg/mL CRISPLD2, or combined with 20 μM MG132 or 10 mM CQ, and TLR4 protein level was assessed by western blotting. Primary hepatocytes treated with 5 μg/mL HMGB1, 10 μg/mL CRISPLD2, or a combination of them. (C) Western blotting analysis of the protein levels of ATG3, ATG7, ATG12, ATG16L1, LC3 II/I, and p62. (D) The protein interaction between TLR4 and LC3 or p62 was confirmed by Co-IP. n=3. One-way ANOVA was performed for statistical analysis. * p <0.05, ** p <0.01, and *** p <0.001. Abbreviations: ANOVA, analysis of variance; ATG, autophagy-related protein; CQ, chloroquine; Co-IP, co-immunoprecipitation; CRISPLD2, cysteine-rich secreted protein LCCL domain 2; HMGB1, high mobility group box 1; LC3, microtubule-associated protein 1 light chain 3; MG132, proteasome inhibitor; TLR4, toll-like receptor 4.

Article Snippet: The primary hepatocytes were treated with 10 nM TLR4 inhibitor (TAK-242, HY-11109; MCE, NJ, USA) for 24 hours; 100 μM palmitoylation inhibitor, 2-bromopalmitate (2-BP, HY-111770; MCE) for 2 and 4 hours; 10 μM palmitoylation enhancer, palmostatin B (HY-120911; MCE) for 2, 4, and 8 hours; 20 μM proteasome inhibitor MG132 (HY-13259; MCE); and 10 mM autophagy–lysosome inhibitor chloroquine (CQ, HY-17589A; MCE) for 4 hours.

Techniques: Western Blot, Co-Immunoprecipitation Assay, Immunoprecipitation

Journal: Hepatology Communications

Article Title: CRISPLD2 protects against liver inflammation and fibrosis via GRP78 to repress HMGB1/TLR4 axis–mediated STING palmitoylation

doi: 10.1097/HC9.0000000000000954

Figure Lengend Snippet: CRISPLD2 interacted with GRP78 to cause TLR4 degradation, and consequently restrained STING palmitoylation–mediated hepatocyte inflammatory response and fibrosis. (A) Western blotting analysis of GRP78 protein level in primary hepatocytes infected with lentiviruses carrying shNC or shGRP78. CRISPLD2-treated primary hepatocytes were infected with lentiviruses carrying shNC or shGRP78. (B) The binding of CRISPLD2 to GRP78 protein was analyzed by Co-IP. (C, D) The interaction between CRISPLD2 and GRP78 proteins in the cytomembrane was validated by biotin pull-down and Co-IP assay. (E) Immunofluorescent staining observed the subcellular localization of GRP78 in primary hepatocytes (scale bar=25 μm). (F) Western blotting analysis of TLR4, ATG7, LC3 II/I, and p62 protein levels. (G) Co-IP evaluated the binding of TLR4 to LC3 or p62. n=3. The Student t test (for A) and one-way ANOVA (for B–G) were performed for statistical analysis. * p <0.05, ** p <0.01, and *** p <0.001. Abbreviations: ANOVA, analysis of variance; ATG7, autophagy-related protein 7; Co-IP, co-immunoprecipitation; CRISPLD2, cysteine-rich secreted protein LCCL domain 2; GRP78, 78 kDa glucose-regulated protein; LC3, microtubule-associated protein 1 light chain 3; shNC, non-targeting short hairpin RNA control; shGRP78, short hairpin RNA targeting GRP78; STING, stimulator of interferon genes; TLR4, toll-like receptor 4.

Article Snippet: The primary hepatocytes were treated with 10 nM TLR4 inhibitor (TAK-242, HY-11109; MCE, NJ, USA) for 24 hours; 100 μM palmitoylation inhibitor, 2-bromopalmitate (2-BP, HY-111770; MCE) for 2 and 4 hours; 10 μM palmitoylation enhancer, palmostatin B (HY-120911; MCE) for 2, 4, and 8 hours; 20 μM proteasome inhibitor MG132 (HY-13259; MCE); and 10 mM autophagy–lysosome inhibitor chloroquine (CQ, HY-17589A; MCE) for 4 hours.

Techniques: Western Blot, Infection, Binding Assay, Co-Immunoprecipitation Assay, Staining, Immunoprecipitation, shRNA, Control

Journal: Hepatology Communications

Article Title: CRISPLD2 protects against liver inflammation and fibrosis via GRP78 to repress HMGB1/TLR4 axis–mediated STING palmitoylation

doi: 10.1097/HC9.0000000000000954

Figure Lengend Snippet: CRISPLD2 relieved inflammation and liver fibrosis in vivo. CCl 4 -challenged mice were treated with recombinant protein CRISPLD2. (A) Pathological changes in livers were evaluated by HE staining (scale bar=50 μm). (B) Liver fibrosis was examined by the Masson Trichrome and Sirius Red staining (scale bar=50 μm). (C) Serum ALT and AST levels were assessed. (D, E) Immunofluorescent staining evaluated the co-localization of CRISPLD2 and GRP78, TLR4, and LC3, or p62 in liver tissues (scale bar=50 μm). (F) STING palmitoylation level in livers was analyzed by ABE assay. The samples were added with hydroxylamine (+HAM) buffer or lysis buffer (−HAM). Palm-STING, STING palmitoylation. (G) Serum IL-6, IL-1β, and TNF-α levels were determined by ELISA. (H) α-SMA, FN, and col1a1 expression in liver tissues were examined by immunohistochemical staining (scale bar=50 μm). n=6. One-way ANOVA was performed for statistical analysis. * p <0.05, ** p <0.01, and *** p <0.001. Abbreviations: ABE, Acyl-Biotin Exchange; α-SMA, alpha-smooth muscle actin; ANOVA, analysis of variance; ALT, alanine aminotransferase; AST, aspartate aminotransferase; COL1A1, collagen type I alpha 1 chain; CRISPLD2, cysteine-rich secreted protein LCCL domain 2; ELISA, enzyme-linked immunosorbent assay; FN, fibronectin; GRP78, 78 kDa glucose-regulated protein; HAM, hydroxylamine; HE, hematoxylin and eosin; LC3, microtubule-associated protein 1 light chain 3; Palm-STING, STING palmitoylation; STING, stimulator of interferon genes; TLR4, toll-like receptor 4.

Article Snippet: The primary hepatocytes were treated with 10 nM TLR4 inhibitor (TAK-242, HY-11109; MCE, NJ, USA) for 24 hours; 100 μM palmitoylation inhibitor, 2-bromopalmitate (2-BP, HY-111770; MCE) for 2 and 4 hours; 10 μM palmitoylation enhancer, palmostatin B (HY-120911; MCE) for 2, 4, and 8 hours; 20 μM proteasome inhibitor MG132 (HY-13259; MCE); and 10 mM autophagy–lysosome inhibitor chloroquine (CQ, HY-17589A; MCE) for 4 hours.

Techniques: In Vivo, Recombinant, Staining, Lysis, Enzyme-linked Immunosorbent Assay, Expressing, Immunohistochemical staining

Journal: Scientific Reports

Article Title: RBP4 interferes with tongue squamous cell carcinoma progression by inhibiting the PI3K/AKT signaling pathway and promoting macrophage M1-type polarization

doi: 10.1038/s41598-026-39915-4

Figure Lengend Snippet: RBP4 promotes macrophage M1 polarization by activating the TLR4/NF-κB pathway. ( a ): KEGG Bar Charts of human recombinant RBP4 protein-treated M0 macrophage cells after transcriptome sequencing. ( b ): Correlation analysis of RBP4 and TLR4 protein expression using Spearman and Pearson methods. ( c ): Western blotting assay to detect the effect of human recombinant RBP4 on macrophage TLR4/NF-KB pathway and the change of the effect of RBP4 action after the addition of TAK242. ( d , e ): qRT-PCR and western blotting are used to detect the effect of the human recombinant RBP4 protein on the macrophage phenotype and the effect of RBP4 action after the addition of TAK242. ( f , g ): Western blotting experiments are used to detect the effects of changes in RBP4 expression levels on the macrophage TLR4/NF-κB pathway in a Transwell co-culture system. ( h ): Western blotting experiments are used to detect the effects of tumor-secreted RBP4 on the macrophage TLR4/NF-κB pathway after the addition of TAK242. Changes in activation level. ( i ): Western blotting experiments are used to detect the effect of tumor-secreted RBP4 on macrophage phenotype after addition of TAK242 in Transwell co-culture system. TAK242 treatment blocks RBP4 overexpression induced by TSCC cells in the macrophage TLR4/p-NF-κB upregulation and M1-type polarization. KEGG, Kyoto Encyclopedia of Genes and Genomes; TSCC, tongue squamous cell carcinoma. (*Compared with Control, * p < 0.05, ** p < 0.01, *** p < 0.001,*** p < 0.0001).

Article Snippet: Transcriptome sequencing showed that RBP4 activated the TLRs/NF-κB pathway (Fig. a) and that TLR4 and RBP4 expression were positively correlated (Spearman, Fig. b).Western blotting showed elevated expression of TLR4 and p-NF-κB in macrophages incorporating the human recombinant RBP4 protein (with no NF-κB changes) (Fig. c).QRT-PCR and Western blotting confirmed that RBP4 significantly up-regulated M1 markers (CD86, iNOS) and inhibited M2 markers (CD206, Arg-1) (Fig. d, e), whereas the TLR4 inhibitor TAK242 (0.2 Mm, HY-11109, MCE) reversed this effect ( Fig. c, e).

Techniques: Recombinant, Sequencing, Expressing, Western Blot, Quantitative RT-PCR, Co-Culture Assay, Activation Assay, Over Expression, Control

Journal: Scientific Reports

Article Title: RBP4 interferes with tongue squamous cell carcinoma progression by inhibiting the PI3K/AKT signaling pathway and promoting macrophage M1-type polarization

doi: 10.1038/s41598-026-39915-4

Figure Lengend Snippet: Effect of RBP4 on macrophage phenotype. ( a ): Animals were grouped according to the injection of different cells, and the tumor volume, growth curve, and mass were measured and recorded. ( b ): The levels of CD86 and CD206 cell infiltration in the sections of transplanted tumors from each group are analyzed using immunofluorescence staining. ( c ): Changes in the activation level of the TLR4/NF-KB pathway in each group of transplanted tumor tissues are detected using Western blotting. ( d ): Mechanistic diagram of the effects of changes in RBP4 levels on the proliferation, migration, and invasion abilities of TSCC cells and the effects of tumor-secreted RBP4 on the phenotype of TAMs. TAMs, tumor-associated macrophages; TSCC, tongue squamous cell carcinoma. (*Compared with RAW264.7 + Control, * p < 0.05, ** p < 0.01).

Article Snippet: Transcriptome sequencing showed that RBP4 activated the TLRs/NF-κB pathway (Fig. a) and that TLR4 and RBP4 expression were positively correlated (Spearman, Fig. b).Western blotting showed elevated expression of TLR4 and p-NF-κB in macrophages incorporating the human recombinant RBP4 protein (with no NF-κB changes) (Fig. c).QRT-PCR and Western blotting confirmed that RBP4 significantly up-regulated M1 markers (CD86, iNOS) and inhibited M2 markers (CD206, Arg-1) (Fig. d, e), whereas the TLR4 inhibitor TAK242 (0.2 Mm, HY-11109, MCE) reversed this effect ( Fig. c, e).

Techniques: Injection, Immunofluorescence, Staining, Activation Assay, Western Blot, Migration, Control