jc 1 detection  (TargetMol)

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