jc 1 mmp assay kit  (MedChemExpress)

Journal: Bioactive Materials

Article Title: Bioengineered extracellular vesicles escape lysosomal degradation and deliver Tet-PKM2 for macrophage immunometabolic reprogramming and periodontitis treatment

doi: 10.1016/j.bioactmat.2026.01.002

Figure Lengend Snippet: Metabolic reprogramming and enhanced mitochondrial function in LPS-activated macrophages in response to LEV Tet−PKM2 @TA treatment. The macrophages were pretreated with 100 ng/mL LPS for 24 h and then treated with PBS (Control), 100 μg/mL LEVs PKM2 , LEVs Tet−PKM2 , or LEVs Tet−PKM2 @TA for another 24 h. ( A ) Heatmap representing differentially detected metabolites involved in glycolysis and the TCA cycle in the Control, LEVs PKM2 , LEVs Tet−PKM2 , or LEVs Tet−PKM2 @TA groups ( n = 4). ( B ) Concentrations of key glycolysis and TCA metabolites in Control, LEVs PKM2 , LEVs Tet−PKM2 , and LEVs Tet−PKM2 @TA groups ( n = 4). ( C ) Schematic illustration revealing changes in key glycolysis and TCA metabolites in the LEVs Tet−PKM2 @TA group versus the Control group. The up (down) arrows indicate increased (decreased) levels of metabolites in macrophages. ( D ) Kinetic profile of the ECAR in LPS-activated macrophages in response to sequential injections of glucose, oligomycin, and 2-DG in various groups (Seahorse XF test) ( n = 4). ( E ) Quantification of glycolysis, glycolytic capacity and glycolytic reserve in the Control, LEVs PKM2 , LEVs Tet−PKM2 , and LEVs Tet−PKM2 @TA groups ( n = 4). ( F ) Kinetic profile of the OCR in LPS-activated macrophages in response to sequential injections of oligomycin, FCCP, and Rot/AA in various groups (Seahorse XF test) ( n = 4). ( G ) Quantification of basal respiration, ATP production, and maximal respiration in the Control, LEVs PKM2 , LEVs Tet−PKM2 , and LEVs Tet−PKM2 @TA groups ( n = 4). ( H ) JC-1 aggregation (red fluorescence) in healthy mitochondria and cytosolic JC-1 monomers in compromised mitochondria (green fluorescence) (immunofluorescence assays). ( I ) Quantitative analysis of MMP levels determined by the relative ratio of red/green fluorescence intensity in the Control, LEVs PKM2 , LEVs Tet−PKM2 , and LEVs Tet−PKM2 @TA groups ( n = 4). ( J ) Intracellular ATP levels of LPS-activated macrophages in the Control, LEVs PKM2 , LEVs Tet−PKM2 , and LEVs Tet−PKM2 @TA groups ( n = 3). ( K-M ) The macrophages were pretreated with 100 ng/mL LPS for 24 h and then treated with PBS (Control), 10 μM UK-5099, 100 μg/mL LEVs Tet−PKM2 @TA, or 10 μM UK-5099 plus 100 μg/mL LEVs Tet−PKM2 @TA for another 24 h. ( K ) Schematic illustration revealing mechanism of LEVs Tet−PKM2 @TA promotes macrophage metabolic reprogramming depending on pyruvate influx into the TCA cycle. ( L ) Kinetic profile of the OCR in LPS-activated macrophages in response to sequential injections of oligomycin, FCCP, and Rot/AA in various groups (Seahorse XF test) ( n = 3). ( M ) Quantification of basal respiration, ATP production, and maximal respiration in the Control, UK-5099, LEVs Tet−PKM2 @TA, and UK-5099 + LEVs Tet−PKM2 @TA groups ( n = 3). The data are expressed as the mean ± SEM. Statistical analysis was performed with one-way ANOVA ( B , E , G, I, J , and M ). ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 indicate significant differences between the indicated columns.

Article Snippet: The MMP of the macrophages was assessed using a JC-1 MMP Assay Kit (MCE).

Techniques: Control, Fluorescence, Immunofluorescence

Journal: Journal of Nanobiotechnology

Article Title: Targeting TRIM25 as a therapeutic strategy to enhance ferroptosis in glioblastoma cells

doi: 10.1186/s12951-025-03908-8

Figure Lengend Snippet: VDAC2 induces ferroptosis via mitochondrial damage A . GO enrichment analysis of genes differentially expressed between VDAC2-overexpressing cells and parental cells (RNA-seq, n = 3). B . KEGG pathway enrichment analysis of differentially expressed genes in VDAC2-overexpressing versus parental cells. C . Heatmap of VDAC2 expression and ferroptosis-related genes in 6 clinical GBM samples. D . Pearson correlation analysis between VDAC2 and ACSL4/SLC7A11 expression in the TCGA database. E . Viability assessment of live and dead cells under different treatment conditions ( n = 5). Scale bar = 20 μm. F . Flow cytometric quantification of oxidized versus nonoxidized lipid ratios ( n = 5). G . Measurement of intracellular lipid ROS levels by flow cytometry ( n = 5). H . Immunofluorescence imaging of lipid peroxidation using C11-BODIPY probe staining. Scale bar = 10 μm. I . Assessment of changes in the mitochondrial membrane potential (Δψm) via JC-1 probe staining. Scale bar = 10 μm. J . Visualization of mitochondrial lipid peroxidation using MitoPeDPP probe staining. K . Detection of mitochondrial ROS by MitoSOX probe staining. Scale bar = 10 μm. L . Transmission electron microscopy images of cells under different treatments (red arrows indicate mitochondria). M . Tumor volume measurements and H&E staining in orthotopic xenograft models ( n = 8 animals per group). N . Quantitative analysis of tumor growth by fluorescence intensity and volume measurement ( n = 8). O . Survival curves of nude mice bearing tumors derived from parental or VDAC2-overexpressing cells ( n = 8 per group). P . Immunohistochemical (IHC) analysis and quantitative scoring of VDAC2 and 4-HNE expression in tumor tissues ( n = 3). Scale bar = 50 μm. P values were calculated using two-tailed t tests (E, F, G, N and P) and the log-rank Mantel‒Cox test (O). The data are presented as the means ± SDs

Article Snippet: Cells subjected to different treatments were seeded in 6-well plates and stained using a JC-1 detection kit (MCE, USA; HY15534) according to the manufacturer’s protocol.

Techniques: RNA Sequencing, Expressing, Flow Cytometry, Immunofluorescence, Imaging, Staining, Membrane, Transmission Assay, Electron Microscopy, Fluorescence, Derivative Assay, Immunohistochemical staining, Two Tailed Test

Journal: Journal of Nanobiotechnology

Article Title: Targeting TRIM25 as a therapeutic strategy to enhance ferroptosis in glioblastoma cells

doi: 10.1186/s12951-025-03908-8

Figure Lengend Snippet: TRIM25 knockout enhances ferroptosis sensitivity in LN229 cells in vitro and in vivo A . Correlations between TRIM25 expression levels and survival duration in glioma patients across the CGGA, TCGA, and Rembrandt databases. B . Representative immunohistochemical (IHC) staining images of TRIM25 and 4-HNE in recurrent versus nonrecurrent patients from a 180-case clinical tissue microarray cohort. Scale bar = 50 μm. C . Kaplan‒Meier survival analysis evaluating the prognostic significance of TRIM25 expression in glioma patients. D . GO enrichment analysis of differentially expressed genes between TRIM25-knockout and parental cells (RNA-seq, n = 3). E . Gene set enrichment analysis (GSEA) of the ferroptosis pathway. F . Viability assessment of live versus dead cells under different treatment conditions ( n = 5). Scale bar = 20 μm. G . Flow cytometric measurement of intracellular lipid ROS levels ( n = 5). H . Quantitative analysis of oxidized-to-nonoxidized-lipid ratios by flow cytometry ( n = 5). I . Immunofluorescence visualization of lipid peroxidation using C11-BODIPY probe staining. Scale bar = 10 μm. J . Assessment of changes in the mitochondrial membrane potential (Δψm) via JC-1 probe staining. Scale bar = 20 μm. K . Detection of mitochondrial lipid peroxidation using MitoPeDPP probe staining. Scale bar = 10 μm. L . Measurement of mitochondrial ROS by MitoSOX probe staining. Scale bar = 10 μm. M . Tumor volume measurements and H&E staining in orthotopic xenograft models ( n = 8 animals per group). N . Quantitative analysis of tumor growth by fluorescence intensity and volumetric measurements ( n = 8). O . Survival curves of nude mice bearing tumors derived from parental cells, TRIM25 single-knockout cells, or TRIM25/VDAC2 double-knockout cells ( n = 8 animals per group). P . IHC analysis and quantitative scoring of TRIM25, VDAC2, 4-HNE, and ACSL4 expression in tumor tissues ( n = 3). Scale bar = 50 μm. P values were calculated using two-tailed t tests (F, G, H, N and P), the log-rank Mantel‒Cox test (O) and the log-rank test, with adjustments for confounding factors via Cox regression (C). The data are presented as the means ± SDs

Article Snippet: Cells subjected to different treatments were seeded in 6-well plates and stained using a JC-1 detection kit (MCE, USA; HY15534) according to the manufacturer’s protocol.

Techniques: Knock-Out, In Vitro, In Vivo, Expressing, Immunohistochemical staining, Immunohistochemistry, Microarray, RNA Sequencing, Flow Cytometry, Immunofluorescence, Staining, Membrane, Fluorescence, Derivative Assay, Double Knockout, Two Tailed Test

Journal: Journal of Nanobiotechnology

Article Title: Targeting TRIM25 as a therapeutic strategy to enhance ferroptosis in glioblastoma cells

doi: 10.1186/s12951-025-03908-8

Figure Lengend Snippet: Construction and in vitro evaluation of ANP SS (sgTRIM25) A . Schematic of disulfide-crosslinked nanoparticles encapsulating Cas9/sgRNA complexes synthesized via in situ radical polymerization, followed by Angiopep-2 (Ang) functionalization to generate ANP-conjugated ANP SS (sgTRIM25). B . Dynamic light scattering (DLS) analysis showing the particle size distribution of ANP SS (sgTRIM25). C . Zeta potential measurement of ANP SS (sgTRIM25) nanoparticles ( n = 3). D . Fluorescence spectra confirming sulfo-cyanine5.5 (Cy5.5) labeling of ANP SS (sgTRIM25). E . Transmission electron microscopy (TEM) images demonstrating morphological changes in ANP SS (sgTRIM25) in saline with/without GSH. F . Agarose gel electrophoresis showing TRIM25 cleavage patterns by T7 endonuclease I (T7E1) under different treatment conditions. G . Schematic illustration of the genome editing mechanism of ANP SS (sgTRIM25). H . Sanger sequencing analysis identifying precise TRIM25 gene editing sites following ANP SS (sgTRIM25) treatment. I . Immunofluorescence comparison of cellular targeting efficiency between NP SS (sgTRIM25) and ANP SS (sgTRIM25). Scale bar = 10 μm. J . Live/dead cell viability assay after ANP SS (sgTRIM25) treatment ( n = 5). Scale bar = 20 μm. K . Flow cytometric quantification of intracellular lipid ROS levels ( n = 5). L. Flow cytometric analysis of oxidized vs. nonoxidized lipid ratios ( n = 5). M . Immunofluorescence visualization of lipid peroxidation via C11-BODIPY probe staining. Scale bar = 10 μm. N . Assessment of mitochondrial membrane potential (Δψm) changes using JC-1 probe staining. Scale bar = 10 μm. O . Mitochondrial lipid peroxidation was detected through MitoPeDPP probe staining. P . Measurement of mitochondrial reactive oxygen species via MitoSOX probe staining. P values were calculated using two-tailed t tests (J, K and L). The data are presented as the means ± SDs

Article Snippet: Cells subjected to different treatments were seeded in 6-well plates and stained using a JC-1 detection kit (MCE, USA; HY15534) according to the manufacturer’s protocol.

Techniques: In Vitro, Synthesized, In Situ, Zeta Potential Analyzer, Fluorescence, Labeling, Transmission Assay, Electron Microscopy, Saline, Agarose Gel Electrophoresis, Sequencing, Immunofluorescence, Comparison, Viability Assay, Staining, Membrane, Two Tailed Test