tak 242  (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 facilitated the cytomembrane translocation of GRP78. Primary hepatocytes were exposed to 5 μg/mL HMGB1, 10 μg/mL CRISPLD2, or a combination of them. (A) GRP78 protein level was analyzed by western blotting. (B) Interplay between CRISPLD2 and GRP78 proteins was evaluated by Co-IP. (C) The binding of CRISPLD2 to GRP78 protein in the cytomembrane of primary hepatocytes was validated by a biotin pull-down assay. (D) Co-IP validated the interaction between CRISPLD2 and GRP78 proteins in the cytomembrane. (E) The co-localization of CRISPLD2 and GRP78 in CRISPLD2-treated primary hepatocytes was observed by immunofluorescent staining (scale bar=25 μm). (F) The subcellular localization of GRP78 in primary hepatocytes was evaluated by immunofluorescent staining, and the cytomembrane was labeled by WGA staining (scale bar=25 μm). n=3. One-way ANOVA was performed for statistical analysis. Abbreviations: ANOVA, analysis of variance; Co-IP, co-immunoprecipitation; CRISPLD2, cysteine-rich secreted protein LCCL domain 2; GRP78, 78 kDa glucose-regulated protein; HMGB1, high mobility group box 1; WGA, wheat germ agglutinin.

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: Translocation Assay, Western Blot, Co-Immunoprecipitation Assay, Binding Assay, Pull Down Assay, Staining, Labeling, 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: 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: Acta Otorhinolaryngologica Italica

Article Title: Dose-dependent IL-29 activation of TLR4 signalling drives eosinophil infiltration in chronic rhinosinusitis with nasal polyps

doi: 10.14639/0392-100X-A1222

Figure Lengend Snippet: Histological and molecular expression analysis of IL-29 and TLR4 in ECRSwNP. (A) Haematoxylin-eosin staining of the experimental and control groups; magnification 100×; (B) qPCR analysis of IL-29 and TLR4 mRNA expression and their correlation; (C) Correlation analysis of IL-29 and TLR4, showing a positive correlation in the experimental group (r = 0.6018, p < 0.0001); (D) Immunohistochemical expression of IL-29 in the experimental and control groups (D1: control group, 100×; D2: experimental group, 100×; D3: control group, 400×; D4: experimental group, 400×); (E) Immunohistochemical expression of TLR4 in the experimental and control groups (E1: control group, 100×; E2: experimental group, 100×; E3: control group, 400×; E4: experimental group, 400×). qPCR data are presented as mean ± SD, correlation analysis was performed using Pearson correlation coefficient, and p < 0.05 was considered statistically significant. Experimental group n = 30, control group n = 30.

Article Snippet: To evaluate the role of TLR4 in IL-29-mediated eosinophil regulation, a TLR4 blockade group (IL-29 50 ng/mL + TAK-242 10 μM, MedChemExpress, HY-11109) and a TAK-242-only intervention group were established.

Techniques: Expressing, Staining, Control, Immunohistochemical staining

Journal: Acta Otorhinolaryngologica Italica

Article Title: Dose-dependent IL-29 activation of TLR4 signalling drives eosinophil infiltration in chronic rhinosinusitis with nasal polyps

doi: 10.14639/0392-100X-A1222

Figure Lengend Snippet: Effect of IL-29 on TLR4 and downstream signaling molecule activation. (A) Western blot analysis of TLR4 protein expression; (B) Western blot analysis of p-NF-κB protein expression; (C) Western blot analysis of p-MAPK protein expression. Eosinophils were stimulated with different concentrations of IL-29 (10, 25, 50, 100 ng/mL) for 24 hours. β-actin was used as the internal control. Data were obtained from 3 independent experiments. * indicates p < 0.05.

Article Snippet: To evaluate the role of TLR4 in IL-29-mediated eosinophil regulation, a TLR4 blockade group (IL-29 50 ng/mL + TAK-242 10 μM, MedChemExpress, HY-11109) and a TAK-242-only intervention group were established.

Techniques: Activation Assay, Western Blot, Expressing, Control

Journal: Acta Otorhinolaryngologica Italica

Article Title: Dose-dependent IL-29 activation of TLR4 signalling drives eosinophil infiltration in chronic rhinosinusitis with nasal polyps

doi: 10.14639/0392-100X-A1222

Figure Lengend Snippet: The interventional effect of TAK-242 Blocking TLR4 on IL-29 action. (A) Transwell assay showing changes in eosinophil migration after TLR4 blockade with TAK-242; (B) ELISA of IL-5 secretion after TAK-242 intervention; (C) ELISA of IL-13 secretion after TAK-242 intervention; (D) Western blot analysis of TLR4, p-NF-κB, and p-MAPK protein expression after TAK-242 treatment. Data are presented as mean ± SD. * indicates p < 0.05.

Article Snippet: To evaluate the role of TLR4 in IL-29-mediated eosinophil regulation, a TLR4 blockade group (IL-29 50 ng/mL + TAK-242 10 μM, MedChemExpress, HY-11109) and a TAK-242-only intervention group were established.

Techniques: Blocking Assay, Transwell Assay, Migration, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing