tlr9 mediated nf κb reporter assay 244 hek dualtm htlr9  (InvivoGen)

Journal: International Journal of Molecular Sciences

Article Title: Aberrant CX3CL1-CX3CR1 Signaling Reprograms Microglial Exosome Secretion via KIFC2 to Drive Cognitive Impairment in Chronic Pain

doi: 10.3390/ijms27146304

Figure Lengend Snippet: The microglial CX3CL1-CX3CR1 signaling induces neurotoxicity by promoting IL-17-enriched exosome release via the p38 MAPK-NF-κB-KIFC2 axis. ( A , B ) Western blot analysis of p38 MAPK-NF-κB phosphorylation and KIFC2 expression in BV2 cells treated with CX3CL1, in the presence of the p38 inhibitor SB203580 (20 μM) or the NF-κB inhibitor MCCK1 (2 μM). ( C – F ) NTA characterization of EVs secreted by BV2 cells treated with CX3CL1 in combination with SB203580 or MCCK1, displaying representative size distribution profiles ( C ), absolute concentration of small exosomes (<100 nm) ( D ), total EV concentration ( E ), and the relative proportion of exosomes ( F ). ( G ) ELISA quantification of IL-17 concentrations in EVs secreted by BV2 cells following the indicated inhibitor treatments. ( H ) CCK-8 assay assessing the viability of PC12 cells co-cultured with BV2 cells pre-treated with CX3CL1, SB203580, or MCCK1. ( I ) Western blot analysis of PSD95 and cleaved caspase-3 (c-Casp3) expression in PC12 cells co-cultured with similarly pre-treated BV2 cells. p -values were determined by one-way ANOVA followed by Tukey’s post hoc test ( D – H ).

Article Snippet: For pharmacological inhibition, BV2 cells were pre-incubated with either the p38 inhibitor SB203580 (HY-10256, MedChemExpress, 20 μM) or the NF-κB inhibitor MCCK1 (HY-D0162R, MedChemExpress, 2 μM) for 12 h, followed by stimulation with recombinant murine CX3CL1 (RP01393, ABclonal, Woburn, MA, USA) for 24 h. For gene silencing experiments, BV2 cells were transfected with small interfering RNAs (siRNAs) targeting Kifc2 or Cx3cr1 for 36 h, followed by a 24 h stimulation with recombinant CX3CL1.

Techniques: Western Blot, Phospho-proteomics, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Cell Culture

Journal: The Journal of Biological Chemistry

Article Title: Isolinderalactone suppresses the progression of cholangiocarcinoma by modulating the CARMA1-BCL10-MALT1 signalosome

doi: 10.1016/j.jbc.2026.113162

Figure Lengend Snippet: ILL suppresses the NF-κB signaling pathway in CCA cells. A , KEGG pathway enrichment analysis of differentially expressed genes between control and ILL-treated groups. B , GSEA analysis showing enrichment of the NF-κB signaling pathway. C and D , the expressions of p-IκBα (Ser32), IκBα, p-p65 -(Ser468), and p65 in CCA cells treated with different concentrations of ILL were detected by Western blot and quantified (mean ± SD; one-way ANOVA followed by Bonferroni post hoc test; n = 3 biologically independent experiments). E and F , Western blot analysis and quantification of p-IκBα (Ser32), IκBα, p-p65 (Ser468), and p65 in CCA cells treated with ILL, diprovocim, or the combination of ILL and diprovocim (mean ± SD; one-way ANOVA followed by Bonferroni post hoc test; n = 3 biologically independent experiments). G and H , EdU staining and positive rate analysis in CCA cells treated with ILL, diprovocim, or the combination of ILL and diprovocim (mean ± SD; one-way ANOVA followed by Bonferroni post hoc test; n = 3 biologically independent experiments). Scale bars represent 200 μm. I and J , colony formation assays and quantification of colony numbers in CCA cells (mean ± SD; one-way ANOVA followed by Bonferroni post hoc test; n = 3 biologically independent experiments). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: HCCC9810 and RBE cells were pre-exposed to Diprovocim (5 nM, 2 h) and subsequently co-incubated with ILL (20 μm) for 24 h. Additionally, an independent NF-κB activator, PapRIVto (MCE), was applied at 10 μm for 2 h prior to ILL treatment validate the specificity of the rescue effect.

Techniques: Control, Western Blot, Staining

Journal: The Journal of Biological Chemistry

Article Title: Isolinderalactone suppresses the progression of cholangiocarcinoma by modulating the CARMA1-BCL10-MALT1 signalosome

doi: 10.1016/j.jbc.2026.113162

Figure Lengend Snippet: ILL inhibits NF-κB signaling by modulating BCL10 complex formation and inhibiting its ubiquitination. A , co-IP analysis of interactions between BCL10 and CARMA1 or MALT1 in CCA cells expressing WT BCL10. B , co-IP analysis of interactions between BCL10 and CARMA1 or MALT1 in CCA cells expressing WT BCL10 or mutant BCL10 with the effect of ILL. C and D , ubiquitination of BCL10 in BCL10-WT–expressing or BCL10-mut–expressing CCA cells treated with 0 or 20 μm ILL for 24 h.

Article Snippet: HCCC9810 and RBE cells were pre-exposed to Diprovocim (5 nM, 2 h) and subsequently co-incubated with ILL (20 μm) for 24 h. Additionally, an independent NF-κB activator, PapRIVto (MCE), was applied at 10 μm for 2 h prior to ILL treatment validate the specificity of the rescue effect.

Techniques: Ubiquitin Proteomics, Co-Immunoprecipitation Assay, Expressing, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Isolinderalactone suppresses the progression of cholangiocarcinoma by modulating the CARMA1-BCL10-MALT1 signalosome

doi: 10.1016/j.jbc.2026.113162

Figure Lengend Snippet: ILL suppresses tumor growth and NF-κB signaling in vivo . A , schematic diagram of the experimental design for the xenograft mouse model. B , images of excised tumors from each group at the end of the experiment. C , the progression of tumor volume in each group (mean ± SD; one-way ANOVA followed by Bonferroni post hoc test; n = 5 biologically independent experiments). D , the tumor weights at the end of the experiment (mean ± SD; one-way ANOVA followed by Bonferroni post hoc test; n = 5 biologically independent experiments). E , body weight changes of mice during the period (mean ± SD; one-way ANOVA followed by Bonferroni post hoc test; n = 5 biologically independent experiments). F and G , the expressions of p-IκB, IκB, p-p65, and p65 in tumor tissues from each group were detected by Western blot and quantified (mean ± SD; one-way ANOVA followed by Bonferroni post hoc test; n = 3 biologically independent experiments). H , histological analysis of tumor tissues by H&E staining (Scale bars represent 50.8 μm), and IHC staining for Ki-67 and PCNA (Scale bars represent 12.5 μm). I , co-immunoprecipitation analysis of BCL10 interaction with CARMA1 and MALT1 in tumor lysates. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: HCCC9810 and RBE cells were pre-exposed to Diprovocim (5 nM, 2 h) and subsequently co-incubated with ILL (20 μm) for 24 h. Additionally, an independent NF-κB activator, PapRIVto (MCE), was applied at 10 μm for 2 h prior to ILL treatment validate the specificity of the rescue effect.

Techniques: In Vivo, Western Blot, Staining, Immunohistochemistry, Immunoprecipitation

Journal: Translational Oncology

Article Title: Integrated bioinformatics and experimental validation identifies CLIC6 as a novel tumor suppressor regulating NF-κB signaling and immune microenvironment in nasopharyngeal carcinoma

doi: 10.1016/j.tranon.2026.102802

Figure Lengend Snippet: NF-κB pathway activators reverse the regulatory effects of CLIC6 on malignant biological behavior in nasopharyngeal carcinoma cells and macrophage polarization. (A–C) Western Blot analysis of changes in IκBα and P65 protein phosphorylation levels in 5–8F, HNE-1, and C666–1 cells following co-treatment with NF-κB activator 1 and CLIC6 recombinant protein; (D–F) CCK-8 assay measuring proliferation activity of NPC cell lines under the aforementioned treatment conditions; (G-I) Transwell assay showing changes in invasion capacity of NPC cell lines under the aforementioned treatment conditions; (J-L) qRT-PCR detecting expression levels of M1 macrophage inflammatory markers TNF-α, IL-1β, and IL-6 after co-culture with conditioned medium from NPC cells in each treatment group. *P < 0.05, **P < 0.01.

Article Snippet: NF-κB activator 1 (HY-12315) and IKK16 (HY-13313) were obtained from MedChemExpress (USA).

Techniques: Western Blot, Phospho-proteomics, Recombinant, CCK-8 Assay, Activity Assay, Transwell Assay, Quantitative RT-PCR, Expressing, Co-Culture Assay