Review

tlr4 inhibitor tak 242 (MedChemExpress)

MedChemExpress is a verified supplier

MedChemExpress manufactures this product

About

News

Press Release

Team

Advisors

Partners

Contact

Bioz Stars

Bioz vStars

Buy from Supplier

Structured Review

MedChemExpress tlr4 inhibitor tak 242
Tlr4 Inhibitor Tak 242, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 98/100, based on 683 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/tlr4+inhibitor/Resatorvid/10__1038_slash_s41598___026___67479___w-129-2-8
Average 98 stars, based on 683 article reviews

tlr4 inhibitor tak 242 - by Bioz Stars, 2026-10

98/100 stars

Images

Staining:

Article Title: Neutrophil extracellular trap-borne C3-driven endothelial dysfunction in Klebsiella pneumoniae liver abscess
Article Snippet: .. NETs were stained with 100 nM SYTOX Green (Cat: KGE2503-500, KeyGEN BioTECH, China), and the time-dependent induction of NETs was assessed from 0 to 60 min. To investigate the molecular mechanisms, neutrophils were co-incubated with K. pneumoniae and inhibitors, including TLR4 inhibitor (100 nM TAK-242, Cat: HY-11109, MedChemExpress, USA), PI3K p110α inhibitor (1 μM BYL-719, Cat: HY-15244, MedChemExpress, USA), DNase I (40 U, Cat: D8071, Solarbio, China), and C3 peptide inhibitor AMY-101 acetate (5 μM, Cat: HY-P1717B, MedChemExpress, USA). ..

Article Title: Neutrophil extracellular trap-borne C3-driven endothelial dysfunction in Klebsiella pneumoniae liver abscess.
Article Snippet: .. NETs were stained with 100 nM SYTOX Green (Cat: KGE2503-500, KeyGEN BioTECH, China), and the time-dependent induction of NETs was assessed from 0 to 60 min. To investigate the molecular mechanisms, neutrophils were coincubated with K. pneumoniae and inhibitors, including TLR4 inhibitor (100 nM TAK-242, Cat: HY-11109, MedChemExpress, USA), PI3K p110α inhibitor (1 μM BYL-719, Cat: HY-15244, MedChemExpress, USA), DNase I (40 U, Cat: D8071, Solarbio, China), and C3 peptide inhibitor AMY-101 acetate (5 μM, Cat: HYP1717B, MedChemExpress, USA). ..

Mouse Assay:

Article Title: GRK2-facilitated TLR4 signaling promotes cardiac fibrosis in rheumatic mice.
Article Snippet: Objective: Patients with rheumatoid arthritis (RA) have a much higher prevalence of cardiac dysfunction, which explains the high mortality rate in RA patients despite treatment with anti-arthritic drugs.. This study elucidates a molecular mechanism that causes abnormal activation of cardiac fibroblasts in the inflammatory state of RA through transactivation of canonical TLR4 signaling by GRK2-dependent signaling mechanisms, and which ultimately induces heart disease.. Methods and results: Collagen-induced arthritis (CIA) models were established in mice, and the cardiac function in these CIA mice was dynamically examined using echocardiography.

Image Search Results

TLR4 inhibition supports zeolite-mediated reduction of LPS-induced NF-κB activation in THP-1 Dual cells. THP-1 Dual NF-κB reporter cells were pre-treated with the small-molecule TLR4 inhibitor TAK-242 (5 µM) for 1 h prior to stimulation with lipopolysaccharide (LPS) or combined clinoptilolite zeolite and LPS treatment conditions. DMSO (0.1%) was included as a vehicle control. Data are presented as mean ± SD. Statistical significance was determined using an unpaired Student’s t-test. ns, not significant.

Journal: Frontiers in Immunology

Article Title: Adsorption-mediated modulation of lipopolysaccharide bioactivity by clinoptilolite zeolite with in vitro immunomodulatory effects and in vivo safety evaluation

doi: 10.3389/fimmu.2026.1818740

Figure Lengend Snippet: TLR4 inhibition supports zeolite-mediated reduction of LPS-induced NF-κB activation in THP-1 Dual cells. THP-1 Dual NF-κB reporter cells were pre-treated with the small-molecule TLR4 inhibitor TAK-242 (5 µM) for 1 h prior to stimulation with lipopolysaccharide (LPS) or combined clinoptilolite zeolite and LPS treatment conditions. DMSO (0.1%) was included as a vehicle control. Data are presented as mean ± SD. Statistical significance was determined using an unpaired Student’s t-test. ns, not significant.

Article Snippet: To inhibit TLR4 signaling, cells were pre-treated with the small-molecule TLR4 inhibitor TAK-242 ( ) (MedChemExpress, HY-11109) at a final concentration of 5 μM for 1 h prior to stimulation.

Techniques: Inhibition, Activation Assay, Control

$SAA1 activates the TLR4/NF-κB pathway to drive osimertinib resistance. A Heatmap showing the expression patterns of NF-κB-associated genes in PC-9 cells treated with exogenous SAA1 (100 ng/ml, 24 h) compared with PBS. B GSEA plot indicating enrichment of the NF-κB signaling pathway in SAA1-treated PC-9 cells. C - D Western blot analysis of whole-cell lysates ( C , assessing p65, p-p65, IκBα, and p-IκBα) and subcellular fractions ( D , assessing nuclear and cytoplasmic p65) in PC-9 OR and H1975 OR cells transfected with vector control, SAA1 overexpression, SAA1 knockout, or SAA1 knockout rescued with exogenous SAA1. E - F Western blot evaluating the effect of the NF-κB inhibitor BAY 11-7082 (5 µM) on SAA1-induced pathway activation in whole-cell lysates ( E ) and subcellular fractions ( F ). G - H Western blot evaluating the effect of the TLR4 inhibitor TAK-242 (10 µM) on SAA1-induced pathway activation in whole-cell lysates ( G ) and subcellular fractions ( H ). I - L Cell proliferation curves ( I , K ) and relative cell viability assays determining osimertinib IC50 ( J , L ) in PC-9 OR and H1975 OR cells treated with PBS or exogenous SAA1, in the presence or absence of BAY 11-7082 (5 µM) or TAK-242 (10 µM) ( n = 3). Two-way ANOVA tests were applied for panels I - L . Abbreviations: SAA1-OE, SAA1 overexpression; SAA1-KO, SAA1 knockout; OR, osimertinib-resistant; NF-κBi, NF-κB inhibitor; TLR4i, TLR4 inhibitor$

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: ERK/GATAD2A/SAA1 axis drives osimertinib resistance via NF-κB and macrophage reprogramming in EGFR-mutated lung adenocarcinoma

doi: 10.1186/s13046-026-03792-6

Figure Lengend Snippet: SAA1 activates the TLR4/NF-κB pathway to drive osimertinib resistance. A Heatmap showing the expression patterns of NF-κB-associated genes in PC-9 cells treated with exogenous SAA1 (100 ng/ml, 24 h) compared with PBS. B GSEA plot indicating enrichment of the NF-κB signaling pathway in SAA1-treated PC-9 cells. C - D Western blot analysis of whole-cell lysates ( C , assessing p65, p-p65, IκBα, and p-IκBα) and subcellular fractions ( D , assessing nuclear and cytoplasmic p65) in PC-9 OR and H1975 OR cells transfected with vector control, SAA1 overexpression, SAA1 knockout, or SAA1 knockout rescued with exogenous SAA1. E - F Western blot evaluating the effect of the NF-κB inhibitor BAY 11-7082 (5 µM) on SAA1-induced pathway activation in whole-cell lysates ( E ) and subcellular fractions ( F ). G - H Western blot evaluating the effect of the TLR4 inhibitor TAK-242 (10 µM) on SAA1-induced pathway activation in whole-cell lysates ( G ) and subcellular fractions ( H ). I - L Cell proliferation curves ( I , K ) and relative cell viability assays determining osimertinib IC50 ( J , L ) in PC-9 OR and H1975 OR cells treated with PBS or exogenous SAA1, in the presence or absence of BAY 11-7082 (5 µM) or TAK-242 (10 µM) ( n = 3). Two-way ANOVA tests were applied for panels I - L . Abbreviations: SAA1-OE, SAA1 overexpression; SAA1-KO, SAA1 knockout; OR, osimertinib-resistant; NF-κBi, NF-κB inhibitor; TLR4i, TLR4 inhibitor

Article Snippet: Osimertinib (T2490), gefitinib (T1181), afatinib ( T21312 ), the NF-κB inhibitor BAY 11-7082 (T1902), the TLR2 inhibitor C29 (T3502), the TLR4 inhibitor TAK-242 (TQ0181), the ERK inhibitor SCH772984 (T6066), and recombinant human SAA1 (TMPJ-00480) were purchased from TargetMol (Boston, MA, USA).

Techniques: Expressing, Western Blot, Analysis, Transfection, Plasmid Preparation, Control, Over Expression, Knock-Out, Activation Assay

SAA1 reshapes the tumor microenvironment by promoting macrophage recruitment and pro-tumorigenic polarization. A - B Representative immunofluorescence images ( A ) and corresponding quantification ( B ) of F4/80⁺ (green) and CD206⁺ (yellow) macrophages in tumors from PC-9 OR CDX models treated with vehicle or osimertinib, comparing vector control and SAA1-KO groups. Scale bar, 20 μm. C - D Flow cytometry plots ( D ) and statistical quantification ( C ) of M2 macrophage polarization (CD206⁺) in BMDM and THP-1 cells treated with PBS or exogenous SAA1 (100 ng/ml, 24 h). E qPCR analysis of classical M2 macrophage markers ( CD206 , VEGFA , TGFB1 , IL-10 , ARG1 ) in THP-1 and BMDM cells co-cultured with PC-9 OR cell lines under osimertinib treatment, comparing vector control and SAA1 knockout conditions ( n = 3). F qPCR analysis of newly proposed macrophage markers ( SPP1 , C1QC , ISG15 , FN1 ) in THP-1 and BMDM cells after exposure to exogenous SAA1(100 ng/ml, 24 h) ( n = 3). G qPCR analysis of M2 markers and SPP1 in THP-1 and BMDM cells treated with TAK-242 (10 µM, 24 h), with or without exogenous SAA1 (100 ng/ml, 24 h) ( n = 3). H - I Flow cytometry analysis ( H ) and comprehensive quantification ( I ) of CD206⁺ macrophages in THP-1 cells treated with PBS or exogenous SAA1 (100 ng/ml, 24 h), combined with DMSO, TAK-242 (10 µM, 24 h), or C29 (50 µM, 24 h), demonstrating the comparative blockade efficacy of distinct TLR pathways ( n = 3). J - L Representative multiplex immunofluorescence images ( L ) and quantitative analysis ( J , K ) of CD68⁺ (red), CD206⁺ (green), and SPP1⁺ (yellow) macrophage infiltration in paired LUAD patient samples before (TKI-naïve) and after (TKI-residual) osimertinib therapy. Scale bar, 50 μm. Student’s t-tests were for panels C , F , J , K and one-way or two-way ANOVA with Dunnett’s multiple comparison tests were for panels B, E, G, I. Abbreviations: OR, osimertinib-resistant; SAA1-KO, SAA1 knockout; Exo, exogenous; TKI, tyrosine kinase inhibitor; IHC, immunohistochemistry

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: ERK/GATAD2A/SAA1 axis drives osimertinib resistance via NF-κB and macrophage reprogramming in EGFR-mutated lung adenocarcinoma

doi: 10.1186/s13046-026-03792-6

Figure Lengend Snippet: SAA1 reshapes the tumor microenvironment by promoting macrophage recruitment and pro-tumorigenic polarization. A - B Representative immunofluorescence images ( A ) and corresponding quantification ( B ) of F4/80⁺ (green) and CD206⁺ (yellow) macrophages in tumors from PC-9 OR CDX models treated with vehicle or osimertinib, comparing vector control and SAA1-KO groups. Scale bar, 20 μm. C - D Flow cytometry plots ( D ) and statistical quantification ( C ) of M2 macrophage polarization (CD206⁺) in BMDM and THP-1 cells treated with PBS or exogenous SAA1 (100 ng/ml, 24 h). E qPCR analysis of classical M2 macrophage markers ( CD206 , VEGFA , TGFB1 , IL-10 , ARG1 ) in THP-1 and BMDM cells co-cultured with PC-9 OR cell lines under osimertinib treatment, comparing vector control and SAA1 knockout conditions ( n = 3). F qPCR analysis of newly proposed macrophage markers ( SPP1 , C1QC , ISG15 , FN1 ) in THP-1 and BMDM cells after exposure to exogenous SAA1(100 ng/ml, 24 h) ( n = 3). G qPCR analysis of M2 markers and SPP1 in THP-1 and BMDM cells treated with TAK-242 (10 µM, 24 h), with or without exogenous SAA1 (100 ng/ml, 24 h) ( n = 3). H - I Flow cytometry analysis ( H ) and comprehensive quantification ( I ) of CD206⁺ macrophages in THP-1 cells treated with PBS or exogenous SAA1 (100 ng/ml, 24 h), combined with DMSO, TAK-242 (10 µM, 24 h), or C29 (50 µM, 24 h), demonstrating the comparative blockade efficacy of distinct TLR pathways ( n = 3). J - L Representative multiplex immunofluorescence images ( L ) and quantitative analysis ( J , K ) of CD68⁺ (red), CD206⁺ (green), and SPP1⁺ (yellow) macrophage infiltration in paired LUAD patient samples before (TKI-naïve) and after (TKI-residual) osimertinib therapy. Scale bar, 50 μm. Student’s t-tests were for panels C , F , J , K and one-way or two-way ANOVA with Dunnett’s multiple comparison tests were for panels B, E, G, I. Abbreviations: OR, osimertinib-resistant; SAA1-KO, SAA1 knockout; Exo, exogenous; TKI, tyrosine kinase inhibitor; IHC, immunohistochemistry

Techniques: Immunofluorescence, Plasmid Preparation, Control, Flow Cytometry, Analysis, Cell Culture, Knock-Out, Multiplex Assay, Comparison, Immunohistochemistry, Co-Culture

TLR2/TBK1 signaling drives IL-33 mRNA expression. (A) IL-33 mRNA expression in cells stimulated with M. genitalium (100 MOI), heat-killed M. genitalium (HK-Mg, equivalent to MOI 100), or purified membrane lipoproteins (LP, 10μg/ml). (B) Cells were pretreated with anti-TLR2 Ab (10μg/ml), C29 (50μmol/L), or TAK-242 (1μmol/L) for 1h prior to infection; IL-33 levels were measured by qPCR. DMSO (0.1%) employed as the vehicle control. (C) Immunoblot of p-TBK1 (Ser172) and total TBK1 kinetics following infection. (D) Cells were treated with the TBK1 inhibitor BX795 (10 μmol/L) and IL-33 mRNA was quantified by qPCR. (E) End1/E6E7 cells were transfected with TBK1 siRNA, and the knockdown efficiency was assessed by qPCR. (F) IL-33 mRNA levels were measured in TBK1−depleted cells by qPCR. (G) Co-IP of TLR2 and TBK1 from cell lysates infected with M. genitalium . Data are mean ± SEM of three independent biological experiments (n=3). *p < 0.05, **p < 0.01 as compared with the uninfected control (A) or indicated groups (B, D–G) . ns, no significant difference between groups (B, E) .

Journal: Frontiers in Immunology

Article Title: Nuclear IL-33 amplifies NF-κB signaling to drive neutrophil recruitment and activation in the endocervical epithelium during Mycoplasma genitalium infection

doi: 10.3389/fimmu.2026.1820874

Figure Lengend Snippet: TLR2/TBK1 signaling drives IL-33 mRNA expression. (A) IL-33 mRNA expression in cells stimulated with M. genitalium (100 MOI), heat-killed M. genitalium (HK-Mg, equivalent to MOI 100), or purified membrane lipoproteins (LP, 10μg/ml). (B) Cells were pretreated with anti-TLR2 Ab (10μg/ml), C29 (50μmol/L), or TAK-242 (1μmol/L) for 1h prior to infection; IL-33 levels were measured by qPCR. DMSO (0.1%) employed as the vehicle control. (C) Immunoblot of p-TBK1 (Ser172) and total TBK1 kinetics following infection. (D) Cells were treated with the TBK1 inhibitor BX795 (10 μmol/L) and IL-33 mRNA was quantified by qPCR. (E) End1/E6E7 cells were transfected with TBK1 siRNA, and the knockdown efficiency was assessed by qPCR. (F) IL-33 mRNA levels were measured in TBK1−depleted cells by qPCR. (G) Co-IP of TLR2 and TBK1 from cell lysates infected with M. genitalium . Data are mean ± SEM of three independent biological experiments (n=3). *p < 0.05, **p < 0.01 as compared with the uninfected control (A) or indicated groups (B, D–G) . ns, no significant difference between groups (B, E) .

Article Snippet: Specific pharmacological inhibitors included: the TLR2 inhibitor C29 (MedChemExpress); the TLR4 signaling inhibitor TAK-242 (CLI-095, InvivoGen); and the specific TBK1/IKKε inhibitor BX795 (InvivoGen).

Techniques: Expressing, Purification, Membrane, Infection, Control, Western Blot, Transfection, Knockdown, Co-Immunoprecipitation Assay

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).

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

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).

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

( A to D ) Monocyte-derived dendritic cells (MoDCs) were treated with NET supernatants formed by PBS, ionomycin high dose (1 μM, Iono-High), native S. Parasanguinis (SP) lysates with low dose of ionomycin (0.01 μM), or SPI (native S. parasanguinis lysates with low-dose ionomycin) with PAD inhibitor (PADi) or intact citrullinated S. parasanguinis lysates, or SPI NETs in the presence or absence of TLR4 inhibitor TAK-242, or IRAK4 inhibitor zimlovisertib for 12 hr, and visualized to detect phagocytosed citrullinated proteins (A), or the level of TLR4 and MyD88 (B) in MoDCs by confocal microscopy. Relative mean intensity of cit-H3 (A, right , n = 4), TLR4, or MyD88 (B, n = 4) was determined with image J. Scale bars, 10 μm. The expression level of TLR4 (C, n = 6) or IRAK4 (D, n = 6) in CD11c + MoDCs was assessed by flow cytometry. ( E and F ) HEK-Blue-hTLR4 cells were treated with each NET supernatant (E, n = 9) or native (Nat) or citrullinated (Cit) human proteins in different concentrations (F, n = 3) for 24 hr. The level of secreted embryonic alkaline phosphatase (SEAP) activity was measured in the supernatant. SPI; S. parasanguinis lysates with low-dose ionomycin. Statistical analysis was determined using one-way ANOVA with Tukey’s multiple comparisons test (B-F). Data are plotted as means ± SEM. *P < 0.05. **P < 0.01, ***P < 0.001.

Journal: bioRxiv

Article Title: Cross-presentation of citrullinated antigens drives cytotoxic CD8 + T cell responses in rheumatoid arthritis

doi: 10.64898/2026.06.03.729882

Figure Lengend Snippet: ( A to D ) Monocyte-derived dendritic cells (MoDCs) were treated with NET supernatants formed by PBS, ionomycin high dose (1 μM, Iono-High), native S. Parasanguinis (SP) lysates with low dose of ionomycin (0.01 μM), or SPI (native S. parasanguinis lysates with low-dose ionomycin) with PAD inhibitor (PADi) or intact citrullinated S. parasanguinis lysates, or SPI NETs in the presence or absence of TLR4 inhibitor TAK-242, or IRAK4 inhibitor zimlovisertib for 12 hr, and visualized to detect phagocytosed citrullinated proteins (A), or the level of TLR4 and MyD88 (B) in MoDCs by confocal microscopy. Relative mean intensity of cit-H3 (A, right , n = 4), TLR4, or MyD88 (B, n = 4) was determined with image J. Scale bars, 10 μm. The expression level of TLR4 (C, n = 6) or IRAK4 (D, n = 6) in CD11c + MoDCs was assessed by flow cytometry. ( E and F ) HEK-Blue-hTLR4 cells were treated with each NET supernatant (E, n = 9) or native (Nat) or citrullinated (Cit) human proteins in different concentrations (F, n = 3) for 24 hr. The level of secreted embryonic alkaline phosphatase (SEAP) activity was measured in the supernatant. SPI; S. parasanguinis lysates with low-dose ionomycin. Statistical analysis was determined using one-way ANOVA with Tukey’s multiple comparisons test (B-F). Data are plotted as means ± SEM. *P < 0.05. **P < 0.01, ***P < 0.001.

Article Snippet: B cells were pre-treated with pharmacologic inhibitors, including 5 μM TLR4 inhibitor, 5 μM E6446 TLR7 inhibitor (Sellekchem), 5 μM IRKA4 inhibitor, or 1 μM Evobrutinib BTK inhibitor (MedChemExpress) 30 min before stimulation of B cells.

Techniques: Derivative Assay, Confocal Microscopy, Expressing, Flow Cytometry, Activity Assay

( A ) Experimental schematic of coculture assay of in vitro MoDCs and CD8 + T cells from ACPA + RA PBMCs in presence of NETs or human citrullinated proteins with or without TLR4 or IRAK4 inhibitor, or anti-HLA class I antibody. Figure created with BioRender. ( B ) Quantification of GZMB + IFNγ + ( left , n = 7), or CD107a + ( right , n = 7) CD8 + T cells in the coculture assay measured by flow cytometry. ( C ) Representative flow cytometry histograms ( left ), and frequency ( right ) of proliferating CD8 + T cells gating with cell proliferation dye eF450 ( n = 6). ( D ) Percentage of IL-6-expressing CD11c + MoDCs in the coculture assay ( n = 5). ( E and F ) Human native- or citrullinated proteins were incubated with MoDCs in the presence or absence of inhibitors and cocultured with CFSE-stained CD8 + T cells. LPS and anti-CD40 antibody were used as a positive control to activate MoDCs. Proportion of GZMB + IFNγ + (E), or proliferating (F) CD8 + T cells was measured using flow cytometry. * indicates versus Cit Ag (E). n = 8. ( G ) Representative histogram showing TLR4 shRNA knockdown efficiency in MoDCs ( left ) or quantification of GZMB + IFNγ + CD8 + T cells ( right ) in the coculture of control ( red ) or TLR4 ( black ) shRNA-treated MoDCs with autologous CD8 + T cells. Each dot represents an independent sample ( n = 3). SPI; S. parasanguinis lysates with low-dose ionomycin. Statistical analysis was determined using one-way ANOVA with Tukey’s multiple comparisons test (B-F), or two-way ANOVA with Fisher’s LSD test (G). Data are plotted as means ± SEM. *P < 0.05. **P < 0.01, ***P < 0.001.

Journal: bioRxiv

Article Title: Cross-presentation of citrullinated antigens drives cytotoxic CD8 + T cell responses in rheumatoid arthritis

doi: 10.64898/2026.06.03.729882

Figure Lengend Snippet: ( A ) Experimental schematic of coculture assay of in vitro MoDCs and CD8 + T cells from ACPA + RA PBMCs in presence of NETs or human citrullinated proteins with or without TLR4 or IRAK4 inhibitor, or anti-HLA class I antibody. Figure created with BioRender. ( B ) Quantification of GZMB + IFNγ + ( left , n = 7), or CD107a + ( right , n = 7) CD8 + T cells in the coculture assay measured by flow cytometry. ( C ) Representative flow cytometry histograms ( left ), and frequency ( right ) of proliferating CD8 + T cells gating with cell proliferation dye eF450 ( n = 6). ( D ) Percentage of IL-6-expressing CD11c + MoDCs in the coculture assay ( n = 5). ( E and F ) Human native- or citrullinated proteins were incubated with MoDCs in the presence or absence of inhibitors and cocultured with CFSE-stained CD8 + T cells. LPS and anti-CD40 antibody were used as a positive control to activate MoDCs. Proportion of GZMB + IFNγ + (E), or proliferating (F) CD8 + T cells was measured using flow cytometry. * indicates versus Cit Ag (E). n = 8. ( G ) Representative histogram showing TLR4 shRNA knockdown efficiency in MoDCs ( left ) or quantification of GZMB + IFNγ + CD8 + T cells ( right ) in the coculture of control ( red ) or TLR4 ( black ) shRNA-treated MoDCs with autologous CD8 + T cells. Each dot represents an independent sample ( n = 3). SPI; S. parasanguinis lysates with low-dose ionomycin. Statistical analysis was determined using one-way ANOVA with Tukey’s multiple comparisons test (B-F), or two-way ANOVA with Fisher’s LSD test (G). Data are plotted as means ± SEM. *P < 0.05. **P < 0.01, ***P < 0.001.

Techniques: Co-culture Assay, In Vitro, Flow Cytometry, Expressing, Incubation, Staining, Positive Control, shRNA, Knockdown, Control

( A ) Pan CD19 + B cells from ACPA + RA PBMCs were treated with anti-IgM antibody, human native or citrullinated proteins, with or without the pretreatment of TLR4, TLR7, or IRAK4 inhibitor for 16 hr. Quantification of CD80 ( left ) or CD86 ( right ) in CD19 + B cells was measured using flow cytometry. n = 6. ( B ) Frequencies of CD80 ( left ), and CD86 ( right ) in ACPA-tetramer + or - tetramer − B cells. n = 8. ( C ) The secreted level of ACPA in the supernatants of pan B cells in response to each stimulation was quantified using ACPA ELISA. n = 5. ( D and E ) Percentage of ACPA + B cells in pan B cells stimulated with anti-IgM antibody, LPS, or human native or citrullinated proteins in the presence or absence of each inhibitor. D; n = 5, E; n = 9. ( F ) Quantification of GZMB + IFNγ + ( left, n = 10) or proliferating ( right, n = 7) CD8 + T cells in the coculture of ACPA + or ACPA − B cells with cell proliferation dye-stained CD8 + T cells with human native- or citrullinated proteins. ( G ) Frequencies of CD86 + ( left ) or IL-6 + ( right ) CD19 + B cells in the coculture of FACS-sorted ACPA + or ACPA − B cells with CD8 + T cells. n = 10. Statistical analysis was determined using one-way ANOVA with Tukey’s multiple comparisons test (A, C-G), or Student’s unpaired t-test (B). Data are plotted as means ± SEM. *P < 0.05. **P < 0.01, ***P < 0.001.

Journal: bioRxiv

Article Title: Cross-presentation of citrullinated antigens drives cytotoxic CD8 + T cell responses in rheumatoid arthritis

doi: 10.64898/2026.06.03.729882

Figure Lengend Snippet: ( A ) Pan CD19 + B cells from ACPA + RA PBMCs were treated with anti-IgM antibody, human native or citrullinated proteins, with or without the pretreatment of TLR4, TLR7, or IRAK4 inhibitor for 16 hr. Quantification of CD80 ( left ) or CD86 ( right ) in CD19 + B cells was measured using flow cytometry. n = 6. ( B ) Frequencies of CD80 ( left ), and CD86 ( right ) in ACPA-tetramer + or - tetramer − B cells. n = 8. ( C ) The secreted level of ACPA in the supernatants of pan B cells in response to each stimulation was quantified using ACPA ELISA. n = 5. ( D and E ) Percentage of ACPA + B cells in pan B cells stimulated with anti-IgM antibody, LPS, or human native or citrullinated proteins in the presence or absence of each inhibitor. D; n = 5, E; n = 9. ( F ) Quantification of GZMB + IFNγ + ( left, n = 10) or proliferating ( right, n = 7) CD8 + T cells in the coculture of ACPA + or ACPA − B cells with cell proliferation dye-stained CD8 + T cells with human native- or citrullinated proteins. ( G ) Frequencies of CD86 + ( left ) or IL-6 + ( right ) CD19 + B cells in the coculture of FACS-sorted ACPA + or ACPA − B cells with CD8 + T cells. n = 10. Statistical analysis was determined using one-way ANOVA with Tukey’s multiple comparisons test (A, C-G), or Student’s unpaired t-test (B). Data are plotted as means ± SEM. *P < 0.05. **P < 0.01, ***P < 0.001.

Techniques: Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining

Active secretion of HMGB1 during taxane treatment is dependent upon TLR4 (A) Flow cytometric detection of intracellular HMGB1 within live PyMT-B6 cells 24 h post-chemotherapy treatment. Data reflect mean ± SD of three biological replicates (right), with significance determined by one-way ANOVA with Sidak’s test for multiple comparisons, shown as ∗ p < 0.1 and ∗∗∗ p < 0.001. One of three representative experiments shown. (B) Confocal microscopy images (left) displaying HMGB1 (green) and DNA (DAPI, blue) in PyMT-B6 cells pretreated with leptomycin B or cytochalasin B for 1 h prior to DTX treatment. Scale bars, 25 μM. Quantification of nuclear HMGB1 is shown on the right. (C) Confocal microscopy images (left) displaying extracellular staining of LAMP1 (CD107a, orange) and DNA (DAPI, blue) in PyMT-B6 cells pretreated with cytochalasin B for 1 h prior to DTX treatment. Scale bars, 25 μM. Quantification of extracellular LAMP1 is shown on the right. (D) Confocal microscopy images (left) displaying HMGB1 (green) and DNA (DAPI, blue) in PyMT-B6 cells pretreated with TLR4 inhibitor ( M62812 ) for 1 h prior to DTX treatment. Scale bars, 10 μM. Quantification of nuclear HMGB1 is shown on the right. For (B–D), quantification reflects the MFI ± SD from individual cells in 2–3 images, with significance determined by one-way ANOVA with Sidak’s test for multiple comparisons, shown as ∗∗∗ p < 0.001. One of three representative experiments shown. (E) Extracellular flow cytometric detection of LAMP1 in PyMT-B6 cells pretreated with a TLR4 inhibitor ( M62812 ) for 1 h prior to DTX treatment. One of three independent experiments shown. Quantification reflects the MFI ±SD, with significance determined by one-way ANOVA with Sidak’s test for multiple comparisons, shown as ∗ p < 0.05. (F) Flow cytometric analysis of TLR4 expression in non-targeted control (NTC) and Tlr4 -deficient PyMT-B6 cells (top). Flow cytometric detection of nuclear HMGB1 in TLR4-proficient versus TLR4-deficient PyMT-B6 cells following PTX treatment (bottom). One of three independent experiments shown. Quantification reflects the MFI ± SD, with significance determined by one-way ANOVA with Sidak’s test for multiple comparisons, shown as ∗∗∗ p < 0.001. (G) Percentage change in tumor volume in mice bearing orthotopic Tlr4 -deficient PyMT-B6 tumors treated with PTX and either IgG 2a isotype control or αTIM-3. Treatment was initiated when tumors reached ∼50–100 mm 3 (day 0). One of two representative experiments shown with n = 5 mice per group. Data displayed mean ± SEM with significance determined by two-way ANOVA, shown as ∗∗ p < 0.01.

Journal: Cell Reports Medicine

Article Title: Taxane chemotherapy promotes response to TIM-3 checkpoint blockade via STING-mediated ER stress and HMGB1 secretion

doi: 10.1016/j.xcrm.2026.102788

Figure Lengend Snippet: Active secretion of HMGB1 during taxane treatment is dependent upon TLR4 (A) Flow cytometric detection of intracellular HMGB1 within live PyMT-B6 cells 24 h post-chemotherapy treatment. Data reflect mean ± SD of three biological replicates (right), with significance determined by one-way ANOVA with Sidak’s test for multiple comparisons, shown as ∗ p < 0.1 and ∗∗∗ p < 0.001. One of three representative experiments shown. (B) Confocal microscopy images (left) displaying HMGB1 (green) and DNA (DAPI, blue) in PyMT-B6 cells pretreated with leptomycin B or cytochalasin B for 1 h prior to DTX treatment. Scale bars, 25 μM. Quantification of nuclear HMGB1 is shown on the right. (C) Confocal microscopy images (left) displaying extracellular staining of LAMP1 (CD107a, orange) and DNA (DAPI, blue) in PyMT-B6 cells pretreated with cytochalasin B for 1 h prior to DTX treatment. Scale bars, 25 μM. Quantification of extracellular LAMP1 is shown on the right. (D) Confocal microscopy images (left) displaying HMGB1 (green) and DNA (DAPI, blue) in PyMT-B6 cells pretreated with TLR4 inhibitor ( M62812 ) for 1 h prior to DTX treatment. Scale bars, 10 μM. Quantification of nuclear HMGB1 is shown on the right. For (B–D), quantification reflects the MFI ± SD from individual cells in 2–3 images, with significance determined by one-way ANOVA with Sidak’s test for multiple comparisons, shown as ∗∗∗ p < 0.001. One of three representative experiments shown. (E) Extracellular flow cytometric detection of LAMP1 in PyMT-B6 cells pretreated with a TLR4 inhibitor ( M62812 ) for 1 h prior to DTX treatment. One of three independent experiments shown. Quantification reflects the MFI ±SD, with significance determined by one-way ANOVA with Sidak’s test for multiple comparisons, shown as ∗ p < 0.05. (F) Flow cytometric analysis of TLR4 expression in non-targeted control (NTC) and Tlr4 -deficient PyMT-B6 cells (top). Flow cytometric detection of nuclear HMGB1 in TLR4-proficient versus TLR4-deficient PyMT-B6 cells following PTX treatment (bottom). One of three independent experiments shown. Quantification reflects the MFI ± SD, with significance determined by one-way ANOVA with Sidak’s test for multiple comparisons, shown as ∗∗∗ p < 0.001. (G) Percentage change in tumor volume in mice bearing orthotopic Tlr4 -deficient PyMT-B6 tumors treated with PTX and either IgG 2a isotype control or αTIM-3. Treatment was initiated when tumors reached ∼50–100 mm 3 (day 0). One of two representative experiments shown with n = 5 mice per group. Data displayed mean ± SEM with significance determined by two-way ANOVA, shown as ∗∗ p < 0.01.

Article Snippet: M62812 TLR4 inhibitor , Fisher Scientific , Cat# 570510.

Techniques: Confocal Microscopy, Staining, Expressing, Control

Taxanes induce ROS-dependent DNA damage and PARP activation (A) Flow cytometric detection of lipid peroxidation in PyMT-B6 cells pretreated with a TLR4 inhibitor ( M62812 ) or Nox2/4 inhibitor (GLX481304) for 1 h prior to DTX treatment. Quantification on the right reflects the MFI ± SD in live cells from three biological replicates, with significance determined by one-way ANOVA with Sidak’s test for multiple comparisons, shown as ∗∗∗ p < 0.001. One of three independent experiments shown. (B) Confocal microscopy images (left) displaying intracellular staining of HMGB1 (green) and DNA (DAPI, blue) in PyMT-B6 cells pretreated with Nox2/4 inhibitor GLX481304 or N-acetyl cysteine (NAC) for 1 h prior to DTX treatment, with quantification of nuclear HMGB1 (right). Scale bars, 25 μM. (C) Confocal microscopy images (left) displaying interaction of poly-ADP ribosylated chains (PAR) and HMGB1 by proximity ligation assay, with a positive signal indicating proximity of both signals less than 40 nm apart (red), along with DNA staining (DAPI, blue) in PyMT-B6 cells pretreated with a TLR4 inhibitor ( M62812 ), NAC, or Nox2/4 inhibitor (GLX481304) for 1 h prior to DTX treatment for 12 h, with quantification of PARylated HMGB1 (right). Scale bars, 25 μM. (D) Confocal microscopy images (left) displaying HMGB1 (green) and DNA (DAPI, blue) in PyMT-B6 cells pretreated with PARP1/2 inhibitors (niraparib, olaparib, or rucaparib) for 1 h prior to DTX treatment for 24 h, with quantification of nuclear HMGB1 (right). Scale bars, 25 μM. For (B–D), quantification reflects the MFI ± SD in individual cells from 2 to 3 images, with significance determined by one-way ANOVA with Sidak’s test for multiple comparisons, shown as ∗∗∗ p < 0.001. One of three independent experiments shown. (E) Intracellular flow cytometric detection (left) of phosphorylated H2AX (γH2AX) in PyMT cells pretreated with a TLR4 inhibitor ( M62812 ) or NAC for 1 h prior to DTX treatment for 24 h. H 2 O 2 was added as a positive control for staining. Data reflect the mean ± SD of three biological replicates (right), with one representative experiment of three shown. Significance determined by one-way ANOVA with Sidak’s test for multiple comparisons, shown as ∗∗ p < 0.01 and ∗∗∗ p < 0.001. (F) Confocal microscopy images (left) displaying cytoplasmic dsDNA (green) and DNA (DAPI, blue) in PyMT-B6 cells treated with DTX for 24 h. Scale bars are 25 μM. Quantification reflects dsDNA MFI ± SD in individual cells from 2 to 3 images, with significance determined by one-way ANOVA with Sidak’s test for multiple comparisons, shown as ∗∗∗ p < 0.001. One of three independent experiments shown.

Journal: Cell Reports Medicine

Article Title: Taxane chemotherapy promotes response to TIM-3 checkpoint blockade via STING-mediated ER stress and HMGB1 secretion

doi: 10.1016/j.xcrm.2026.102788

Figure Lengend Snippet: Taxanes induce ROS-dependent DNA damage and PARP activation (A) Flow cytometric detection of lipid peroxidation in PyMT-B6 cells pretreated with a TLR4 inhibitor ( M62812 ) or Nox2/4 inhibitor (GLX481304) for 1 h prior to DTX treatment. Quantification on the right reflects the MFI ± SD in live cells from three biological replicates, with significance determined by one-way ANOVA with Sidak’s test for multiple comparisons, shown as ∗∗∗ p < 0.001. One of three independent experiments shown. (B) Confocal microscopy images (left) displaying intracellular staining of HMGB1 (green) and DNA (DAPI, blue) in PyMT-B6 cells pretreated with Nox2/4 inhibitor GLX481304 or N-acetyl cysteine (NAC) for 1 h prior to DTX treatment, with quantification of nuclear HMGB1 (right). Scale bars, 25 μM. (C) Confocal microscopy images (left) displaying interaction of poly-ADP ribosylated chains (PAR) and HMGB1 by proximity ligation assay, with a positive signal indicating proximity of both signals less than 40 nm apart (red), along with DNA staining (DAPI, blue) in PyMT-B6 cells pretreated with a TLR4 inhibitor ( M62812 ), NAC, or Nox2/4 inhibitor (GLX481304) for 1 h prior to DTX treatment for 12 h, with quantification of PARylated HMGB1 (right). Scale bars, 25 μM. (D) Confocal microscopy images (left) displaying HMGB1 (green) and DNA (DAPI, blue) in PyMT-B6 cells pretreated with PARP1/2 inhibitors (niraparib, olaparib, or rucaparib) for 1 h prior to DTX treatment for 24 h, with quantification of nuclear HMGB1 (right). Scale bars, 25 μM. For (B–D), quantification reflects the MFI ± SD in individual cells from 2 to 3 images, with significance determined by one-way ANOVA with Sidak’s test for multiple comparisons, shown as ∗∗∗ p < 0.001. One of three independent experiments shown. (E) Intracellular flow cytometric detection (left) of phosphorylated H2AX (γH2AX) in PyMT cells pretreated with a TLR4 inhibitor ( M62812 ) or NAC for 1 h prior to DTX treatment for 24 h. H 2 O 2 was added as a positive control for staining. Data reflect the mean ± SD of three biological replicates (right), with one representative experiment of three shown. Significance determined by one-way ANOVA with Sidak’s test for multiple comparisons, shown as ∗∗ p < 0.01 and ∗∗∗ p < 0.001. (F) Confocal microscopy images (left) displaying cytoplasmic dsDNA (green) and DNA (DAPI, blue) in PyMT-B6 cells treated with DTX for 24 h. Scale bars are 25 μM. Quantification reflects dsDNA MFI ± SD in individual cells from 2 to 3 images, with significance determined by one-way ANOVA with Sidak’s test for multiple comparisons, shown as ∗∗∗ p < 0.001. One of three independent experiments shown.

Article Snippet: M62812 TLR4 inhibitor , Fisher Scientific , Cat# 570510.

Techniques: Activation Assay, Confocal Microscopy, Staining, Proximity Ligation Assay, Positive Control

The cGAS-STING pathway is required for HMGB1 release (A) Confocal microscopy images depicting HMGB1 (green) and DNA (DAPI, blue) in sgNTC, cGas- deficient, and Sting- deficient PyMT-B6 cells treated with DTX. Representative images of one of three independent experiments shown (left). Scale bars are 25 μM. Quantification reflects MFI ± SD in individual cells from 2 to 3 images, with significance determined by t test, shown as ∗∗∗ p < 0.001 (right). (B) Tumor volume in mice bearing orthotopic Cgas- or Sting -deficient PyMT-B6 tumors treated with PTX and either IgG 2a isotype control or αTIM-3. Treatment was initiated when tumors reached ∼50–100 mm 3 (day 0). One of two representative experiments shown with n = 5–10 mice per group as indicated. Data displayed as mean ± SEM, with significance determined by two-way ANOVA, shown as ∗∗ p < 0.01. (C) Relative mRNA levels for Cxcl9 , Ifnb1 , and Ifna1 in PyMT-B6 cells treated with DTX or DMXAA, along with blockade of IFNAR1, as determined by RT-PCR. (D) Release of IFN-β measured by ELISA following treatment with DTX, PTX, or DMXAA. For (C and D), data reflect the mean ± SD of three biological replicates from one of two independent experiments, with significance determined by one-way ANOVA and Sidak’s test for multiple comparisons, shown as ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. (E) Western blot of phospho-IRF3 (pIRF3) and IRF3 protein present in PyMT-B6 cells exposed to PTX, Tunicamycin, or DMXAA or DTX ± TLR4 inhibitor as indicated. β-actin used as loading control. Molecular weights in kDa are shown to the left. Representative images from one of two independent experiments shown. (F) Confocal microscopy images (left) depicting the colocalization of the Golgi (red), STING (green), and DNA (DAPI, blue) in control, cGas- deficient, and Sting- deficient PyMT-B6 cells treated with DTX. Scale bars, 25 μM. Quantification reflects the colocalization of STING with the Golgi as determined by Mander’s overlap (top right) and the MFI of STING (bottom right). (G) Confocal microscopy images (left) depicting calreticulin (ER marker, red), STING (green), and DNA (DAPI, blue) in control, cGas- deficient, and Sting- deficient PyMT-B6 cells treated with DTX. Scale bars, 25 μM. Quantification reflects the colocalization of STING with the ER as determined by Mander’s overlap (top right) and the MFI of calreticulin (bottom right). For (F) and (G), data reflect the mean ± SD in individual cells from two to three images, with significance determined by t test and shown as ∗∗ p < 0.01, ∗∗∗ p < 0.001, and one of three independent experiments is shown.

Journal: Cell Reports Medicine

Article Title: Taxane chemotherapy promotes response to TIM-3 checkpoint blockade via STING-mediated ER stress and HMGB1 secretion

doi: 10.1016/j.xcrm.2026.102788

Figure Lengend Snippet: The cGAS-STING pathway is required for HMGB1 release (A) Confocal microscopy images depicting HMGB1 (green) and DNA (DAPI, blue) in sgNTC, cGas- deficient, and Sting- deficient PyMT-B6 cells treated with DTX. Representative images of one of three independent experiments shown (left). Scale bars are 25 μM. Quantification reflects MFI ± SD in individual cells from 2 to 3 images, with significance determined by t test, shown as ∗∗∗ p < 0.001 (right). (B) Tumor volume in mice bearing orthotopic Cgas- or Sting -deficient PyMT-B6 tumors treated with PTX and either IgG 2a isotype control or αTIM-3. Treatment was initiated when tumors reached ∼50–100 mm 3 (day 0). One of two representative experiments shown with n = 5–10 mice per group as indicated. Data displayed as mean ± SEM, with significance determined by two-way ANOVA, shown as ∗∗ p < 0.01. (C) Relative mRNA levels for Cxcl9 , Ifnb1 , and Ifna1 in PyMT-B6 cells treated with DTX or DMXAA, along with blockade of IFNAR1, as determined by RT-PCR. (D) Release of IFN-β measured by ELISA following treatment with DTX, PTX, or DMXAA. For (C and D), data reflect the mean ± SD of three biological replicates from one of two independent experiments, with significance determined by one-way ANOVA and Sidak’s test for multiple comparisons, shown as ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. (E) Western blot of phospho-IRF3 (pIRF3) and IRF3 protein present in PyMT-B6 cells exposed to PTX, Tunicamycin, or DMXAA or DTX ± TLR4 inhibitor as indicated. β-actin used as loading control. Molecular weights in kDa are shown to the left. Representative images from one of two independent experiments shown. (F) Confocal microscopy images (left) depicting the colocalization of the Golgi (red), STING (green), and DNA (DAPI, blue) in control, cGas- deficient, and Sting- deficient PyMT-B6 cells treated with DTX. Scale bars, 25 μM. Quantification reflects the colocalization of STING with the Golgi as determined by Mander’s overlap (top right) and the MFI of STING (bottom right). (G) Confocal microscopy images (left) depicting calreticulin (ER marker, red), STING (green), and DNA (DAPI, blue) in control, cGas- deficient, and Sting- deficient PyMT-B6 cells treated with DTX. Scale bars, 25 μM. Quantification reflects the colocalization of STING with the ER as determined by Mander’s overlap (top right) and the MFI of calreticulin (bottom right). For (F) and (G), data reflect the mean ± SD in individual cells from two to three images, with significance determined by t test and shown as ∗∗ p < 0.01, ∗∗∗ p < 0.001, and one of three independent experiments is shown.

Article Snippet: M62812 TLR4 inhibitor , Fisher Scientific , Cat# 570510.

Techniques: Confocal Microscopy, Control, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot, Marker

STING-dependent ER stress and HMGB1 release in a model of TNBC (A) Confocal microscopy images (left) depicting HMGB1 (green) and DNA (DAPI, blue) in KP1 cells treated with PTX or DTX, with and without TLR4 inhibitor. Scale bars are 25 μM. Representative images of one of three independent experiments shown. Quantification reflects the MFI ± SD in individual cells from 2 to 3 images, with significance determined by t test and shown as ∗∗∗ p < 0.001 (right). (B) Quantification of MFI ± SD in individual cells from two to three confocal microscopy images assaying cytosolic dsDNA post-PTX or -DTX treatment, with significance determined by t test and shown as ∗∗ p < 0.01 and ∗∗∗ p < 0.001. (C) Live cell imaging with ER Tracker Blue-White depicting ER expansion in KP1 cells pretreated with NAC or TLR4 inhibitor for 1 h prior to treatment with DTX. Data reflect the MFI ± SD of three biological replicates. (D) Extracellular flow cytometric detection of LAMP1 on KP1 cells 24 h post-pretreatment with NAC or TLR4 inhibitor for 1 h prior to treatment with DTX. Data reflect the percent positivity ± SD of three biological replicates. For (C and D), significance was determined by one-way ANOVA with Sidak’s test for multiple comparisons, shown as ∗ p < 0.05 and ∗∗∗ p < 0.001. One of three independent experiments shown. (E) Tumor volume in mice bearing orthotopic sgNTC or sg Sting KP1 tumors treated with PTX and either IgG 2a isotype control or αTIM-3. Treatment was initiated when tumors reached ∼50–100 mm 3 (day 0). (F) Tumor volume in mice bearing orthotopic sgNTC or sg Sting KP1 tumors treated with PTX and either IgG 2a isotype control or αPD-1. Treatment was initiated when tumors reached ∼50–100 mm 3 (day 0). (G) Flow cytometric analysis of CD103 + cDC1s and Ly6G + neutrophils in the tumors from (F). Data from one of two independent experiments with n = 5 mice per group. Data shown as mean ± SEM, with significance determined by one-way ANOVA with Sidak’s test for multiple comparisons, shown as ∗ p < 0.05 and ∗∗ p < 0.01. (H) Tumor volume in mice bearing orthotopic sgNTC control or Ifnb1 -deficient KP1 tumors treated with PTX and IgG 2a isotype control or αPD-1. Treatment was initiated when tumors reached ∼50–100 mm 3 (day 0). For (E), (F), and (H), data merged from two independent experiments, with n = 9–10 mice per group. Data shown as mean ± SEM with significance determined by two-way ANOVA displayed with ∗ p < 0.05 and ∗∗ p < 0.01.

Journal: Cell Reports Medicine

Article Title: Taxane chemotherapy promotes response to TIM-3 checkpoint blockade via STING-mediated ER stress and HMGB1 secretion

doi: 10.1016/j.xcrm.2026.102788

Figure Lengend Snippet: STING-dependent ER stress and HMGB1 release in a model of TNBC (A) Confocal microscopy images (left) depicting HMGB1 (green) and DNA (DAPI, blue) in KP1 cells treated with PTX or DTX, with and without TLR4 inhibitor. Scale bars are 25 μM. Representative images of one of three independent experiments shown. Quantification reflects the MFI ± SD in individual cells from 2 to 3 images, with significance determined by t test and shown as ∗∗∗ p < 0.001 (right). (B) Quantification of MFI ± SD in individual cells from two to three confocal microscopy images assaying cytosolic dsDNA post-PTX or -DTX treatment, with significance determined by t test and shown as ∗∗ p < 0.01 and ∗∗∗ p < 0.001. (C) Live cell imaging with ER Tracker Blue-White depicting ER expansion in KP1 cells pretreated with NAC or TLR4 inhibitor for 1 h prior to treatment with DTX. Data reflect the MFI ± SD of three biological replicates. (D) Extracellular flow cytometric detection of LAMP1 on KP1 cells 24 h post-pretreatment with NAC or TLR4 inhibitor for 1 h prior to treatment with DTX. Data reflect the percent positivity ± SD of three biological replicates. For (C and D), significance was determined by one-way ANOVA with Sidak’s test for multiple comparisons, shown as ∗ p < 0.05 and ∗∗∗ p < 0.001. One of three independent experiments shown. (E) Tumor volume in mice bearing orthotopic sgNTC or sg Sting KP1 tumors treated with PTX and either IgG 2a isotype control or αTIM-3. Treatment was initiated when tumors reached ∼50–100 mm 3 (day 0). (F) Tumor volume in mice bearing orthotopic sgNTC or sg Sting KP1 tumors treated with PTX and either IgG 2a isotype control or αPD-1. Treatment was initiated when tumors reached ∼50–100 mm 3 (day 0). (G) Flow cytometric analysis of CD103 + cDC1s and Ly6G + neutrophils in the tumors from (F). Data from one of two independent experiments with n = 5 mice per group. Data shown as mean ± SEM, with significance determined by one-way ANOVA with Sidak’s test for multiple comparisons, shown as ∗ p < 0.05 and ∗∗ p < 0.01. (H) Tumor volume in mice bearing orthotopic sgNTC control or Ifnb1 -deficient KP1 tumors treated with PTX and IgG 2a isotype control or αPD-1. Treatment was initiated when tumors reached ∼50–100 mm 3 (day 0). For (E), (F), and (H), data merged from two independent experiments, with n = 9–10 mice per group. Data shown as mean ± SEM with significance determined by two-way ANOVA displayed with ∗ p < 0.05 and ∗∗ p < 0.01.

Article Snippet: M62812 TLR4 inhibitor , Fisher Scientific , Cat# 570510.

Techniques: Confocal Microscopy, Live Cell Imaging, Control

Review

tlr4 inhibitor tak 242 (MedChemExpress)

Structured Review

Images

Related Articles

Staining:

Mouse Assay:

Similar Products

Image Search Results