crx 527  (InvivoGen)

Journal: Science Advances

Article Title: Complex I inhibition combined with TLR activation in the breast tumor microenvironment educates cytotoxic neutrophils

doi: 10.1126/sciadv.adu5915

Figure Lengend Snippet: MFP injection of 4T1 cells into ( A ) BALB/c or ( D ) SCID-Beige mice. Tumor-bearing mice were treated with vehicle, phenformin, and/or CRX-527 (average tumor volume (mm 3 ) ± SEM; 10 to 12 tumors per group). ( B and E ) Tumor mass for the (B) BALB/c or (E) SCID-Beige cohorts. ( C and F ) Circulating Ly6G+/CD11b+ cells were quantified from tumor-bearing (C) BALB/c or (F) SCID-Beige mice at the endpoint. ( G ) MFP injection of AT3 cells into TLR +/+ or TLR4 −/− mice. Tumor-bearing mice were treated with vehicle, polyIC, and/or phenformin (average tumor volume (mm 3 ) ± SEM; 8 to 10 tumors per group). ( H ) Tumor mass and ( I ) circulating Ly6G+/CD11b+ cells at the endpoint. ( J ) Coculture of control and therapy-entrained neutrophils isolated from TLR +/+ and TLR4 −/− mice with AT3 cells (5:1 ratio) ( n = 4 mice each). E:T, effector:target. ( K ) Relative pNF-κB (Ser 536 ) levels were measured in Ly6G+/CD11b+ circulating neutrophils by flow cytometry (4 to 5 tumor-bearing mice per group). P values were calculated using a one-way ANOVA with a Tukey’s post hoc test.

Article Snippet: The TLR4 agonist (CRX527, tlr4-crx527, InvivoGen) was dissolved in 0.1% triethylamine/dimethyl sulfoxide (DMSO), with three pulses of 30% probe sonication (10 s each) at a concentration of 1 mg/ml.

Techniques: Injection, Control, Isolation, Flow Cytometry

Journal: Science Advances

Article Title: Complex I inhibition combined with TLR activation in the breast tumor microenvironment educates cytotoxic neutrophils

doi: 10.1126/sciadv.adu5915

Figure Lengend Snippet: ( A ) PCA analysis and ( B ) relative enrichment score of proteomics data obtained from 4T1 tumor-infiltrating neutrophils isolated from mice treated with vehicle, phenformin, and/or polyIC ( n = 4 per group). ( C ) Volcano plots showing pairwise comparisons highlighting secretory granule and NADPH oxidase components, S100A8 proteins, and inflammatory mediators that are the most differentially expressed. Heatmap showing the most differentially expressed proteins representing ( D ) granule components or ( E ) the NADPH oxidase machinery. ( F and G ) Relative enrichment score for NF-κB pathway genes within the proteomes of neutrophils isolated from the blood or tumors of 4T1 tumor-bearing mice. Quantification of the number of MPO+ (left) or S100A8+ (right) granules in neutrophils isolated from the bloodstream of ( H ) 4T1 or ( I ) AT3 tumor-bearing mice. For (I), neutrophils were isolated from TLR4 +/+ and TLR4 −/− animals. A minimum of 50 neutrophils were counted among three biological replicates. Representative immunofluorescent images are shown. P values were calculated using a one-way ANOVA with a Tukey’s post hoc test [(H) and (I)].

Article Snippet: The TLR4 agonist (CRX527, tlr4-crx527, InvivoGen) was dissolved in 0.1% triethylamine/dimethyl sulfoxide (DMSO), with three pulses of 30% probe sonication (10 s each) at a concentration of 1 mg/ml.

Techniques: Isolation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: NR4A2 attenuates early brain injury after intracerebral hemorrhage by promoting M2 microglial polarization via TLR4/TRAF6/NF-κB pathway inhibition

doi: 10.1007/s00018-025-05755-0

Figure Lengend Snippet: Screening and identification of the target gene of NR4A2. ( A ) The top 20 enriched KEGG terms identified via RNA sequencing. ( B ) The top 30 Q values of GSEA enrichment. ( C ) Heatmap showing differentially expressed genes (from RNA-seq) between control and NR4A2-overexpressing perihematomal brain tissues. ( D ) RT-qPCR analysis of TLR4 mRNA levels. n = 6. ( E ) Dual luciferase assay. ( F ) Pulled down assay. ( G ) Representative images revealing the colocalization of NR4A2 and TLR4 in the perihematomal brains of ICH-operated rats subjected to vehicle treatment or NR4A2 overexpression. Scale bar, 50 μm. n = 6. ( H ) Co-immunoprecipitation assay. ( I ) ChIP-qPCR assay. ( J ) RT-qPCR analysis of TLR4 mRNA levels after NR4A2 overexpression or knockdown. The data represent the means ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: The TLR4 agonist CRX-527 (0.25 mg/kg; #tlrl-crx527, InvivoGen) was dissolved in DMSO and then intraperitoneally injected before ICH injury, as described in a previous study [ ].

Techniques: RNA Sequencing, Control, Quantitative RT-PCR, Luciferase, Over Expression, Co-Immunoprecipitation Assay, ChIP-qPCR, Knockdown

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: NR4A2 attenuates early brain injury after intracerebral hemorrhage by promoting M2 microglial polarization via TLR4/TRAF6/NF-κB pathway inhibition

doi: 10.1007/s00018-025-05755-0

Figure Lengend Snippet: The TLR4 agonist CRX-527 abolishes NR4A2-dependent BBB protection and the anti-inflammatory M2 phenotype. ( A ) Intercellular junctions were visualized in each group. n = 6. Scale bar, 50 μm. ( B ) The ultrastructure of the BBB in each group was imaged via TEM analysis. n = 6. Scale bar, 250 nm. ( C, D ) The presence of infiltrated IgG. n = 6. Scale bar, 10 μm. ( E ) Quantification of EB extravasation. n = 6. ( F, G ) Relative gene expression of P-selectin ( F ) and ICAM-1 ( G ). ( H , I ) Relative gene expression of CXCL1 ( H ) and CCL2 ( I ). ( J-O ) Representative images of BV2 microglial polarization. n = 6. Scale bar, 50 μm. The data represent the means ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: The TLR4 agonist CRX-527 (0.25 mg/kg; #tlrl-crx527, InvivoGen) was dissolved in DMSO and then intraperitoneally injected before ICH injury, as described in a previous study [ ].

Techniques: Gene Expression

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: NR4A2 attenuates early brain injury after intracerebral hemorrhage by promoting M2 microglial polarization via TLR4/TRAF6/NF-κB pathway inhibition

doi: 10.1007/s00018-025-05755-0

Figure Lengend Snippet: NR4A2 suppresses TLR4-mediated TRAF6/NF-κB inflammatory signals. ( A ) Representative immunoblots and quantification of TLR4, TRAF6, p-NF-κB p65 and NF-κB p65 in each group. n = 6. ( B-D ) Densitometric quantification of ( A ). ( E ) Representative immunofluorescence images showing TLR4 (red), TRAF6 (red) and p-NF-κB p65 (green). ( F – H ) Quantitative analysis of ( E ). n = 6. Scale bar, 50 μm. The data represent the means ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: The TLR4 agonist CRX-527 (0.25 mg/kg; #tlrl-crx527, InvivoGen) was dissolved in DMSO and then intraperitoneally injected before ICH injury, as described in a previous study [ ].

Techniques: Western Blot, Immunofluorescence