jc 1 kit  (Beyotime)

Journal: Advanced Science

Article Title: Carrier‐Free Berberine/Nitidine Chloride Self‐Assembled Nanoparticles Induce Ferroptosis to Overcome Bortezomib Resistance in Multiple Myeloma

doi: 10.1002/advs.77174

Figure Lengend Snippet: Berberine (BBR)/nitidine chloride (NC) self‐assembled nanoparticles (BBR/NC‐SAPs) display potent and selective anti‐myeloma activity by inhibiting proliferation, clonogenicity, DNA synthesis, and inducing mitochondrial dysfunction. (A) Multiple myeloma cells (sensitive: 8226, KMS‐11; bortezomib‐resistant: 8226‐BTZR, KMS‐11‐BTZR) and normal B cells (GM12878) were treated for 48 h with a concentration gradient (0, 2, 4, 6, 8, and 10 µg/mL) of free BBR and NC combination (1:1). (B) Multiple myeloma cells (sensitive: 8226, KMS‐11; bortezomib‐resistant: 8226‐BTZR, KMS‐11‐BTZR) and normal B cells (GM12878) were treated for 48 h with a concentration gradient (0, 2, 4, 6, 8, and 10 µg/mL) of BBR/NC‐SAPs. (C) Cells were treated with the indicated concentrations (0, 2, 4, 6, 8, and 10 µg/mL) of BBR/NC‐SAPs. Colony formation assay demonstrating the potent, dose‐dependent inhibition of clonogenic survival. (D) Cells were treated with the indicated concentrations (0, 2, 4, 6, 8, and 10 µg/mL) of BBR/NC‐SAPs. Analysis of DNA synthesis by EdU incorporation assay utilizing flow cytometry. The percentage of EdU‐positive (green) cells decreased in a dose‐dependent manner upon treatment. (E) Assessment of mitochondrial membrane potential by JC‐1 staining employing flow cytometry. Treatment with BBR/NC‐SAPs induced a dose‐dependent loss of mitochondrial membrane potential. Data are presented as the mean ± SD from three independent experiments. Statistical significance was determined by a one‐way ANOVA followed by Dunnett's multiple comparison test versus the control group. * p < 0.05, ** p < 0.01.

Article Snippet: The mitochondrial membrane potential (ΔΨm) was assessed utilizing the fluorescent probe JC‐1 (MCE, USA) according to the manufacturer's protocol.

Techniques: Activity Assay, DNA Synthesis, Concentration Assay, Colony Assay, Inhibition, Flow Cytometry, Membrane, Staining, Comparison, Control

Journal: Advanced Science

Article Title: Carrier‐Free Berberine/Nitidine Chloride Self‐Assembled Nanoparticles Induce Ferroptosis to Overcome Bortezomib Resistance in Multiple Myeloma

doi: 10.1002/advs.77174

Figure Lengend Snippet: NR2F2 is essential for berberine (BBR)/nitidine chloride (NC) self‐assembled nanoparticles (BBR/NC‐SAPs)‐induced ferroptosis in multiple myeloma (MM) cells. (A) Cytotoxicity assays showing that NR2F2‐knockout cells locked the survival threshold and abolished the dosage‐dependent cytotoxicity of BBR/NC‐SAPs as compared to the control. (B) Colony formation assays revealing that NR2F2 knockout preserves clonogenic potential after BBR/NC‐SAPs treatment. (C) Analysis of DNA synthesis by EdU incorporation assay utilizing flow cytometry, demonstrating that NR2F2 knockout preserves cell proliferation potential after BBR/NC‐SAPs treatment. (D) Assessment of mitochondrial membrane potential (ΔΨm) via JC‐1 or similar dye, denoting that NR2F2 deletion prevents membrane depolarization triggered by BBR/NC‐SAPs. (E) Quantification of intracellular Fe 2+ showing blocked iron accumulation in NR2F2‐deficient cells upon BBR/NC‐SAPs treatment. (F) Lipid ROS levels measured by flow cytometry demonstrating that BBR/NC‐SAPs fail to elevate lipid peroxidation in NR2F2‐knockout cells. Ferroptosis‐related protein levels measured by western blot showing that BBR/NC‐SAPs fail to elevate lipid peroxidation in NR2F2‐knockout cells (G, 8226 and 8226‐BTZR; H, KMS‐11 and KMS‐11‐BTZR).

Article Snippet: The mitochondrial membrane potential (ΔΨm) was assessed utilizing the fluorescent probe JC‐1 (MCE, USA) according to the manufacturer's protocol.

Techniques: Knock-Out, Control, DNA Synthesis, Flow Cytometry, Membrane, Western Blot

Journal: Cell Insight

Article Title: GPAT4 regulates antitumor immune response through activating the mtDNA-cGAS axis in cancer cells

doi: 10.1016/j.cellin.2026.100343

Figure Lengend Snippet: Gpat4 deficiency drives tumor-cell innate immune activation through the mtDNA-cGAS-dependent axis independently of the canonical cataly tic motif. (A) Schematic of glycerolipid and ether-lipid biosynthetic pathways highlighting GPAT4 and related enzymes used for comparison. (B) qPCR analysis of Ifnb1 , Ifi44 and Cxcl10 after depletion of the indicated lipid-metabolic regulators. (C) Domain organization of wild-type GPAT4 and the HXXXXD catalytic-motif mutant used for rescue experiments. (D) qPCR analysis of interferon-responsive genes in Gpat4 -depleted cells reconstituted with empty vector, wild-type GPAT4 or catalytic-mutant GPAT4 to determine whether the immune phenotype depends on the canonical catalytic motif. (E) qPCR analysis of Ifi44 and Ifit1 in control and Gpat4 -depleted cells treated with vehicle or the cGAS inhibitor RU521 to test cGAS dependence. (F) Measurements of mitochondrial membrane potential and reactive oxygen species in control and Gpat4 -depleted cells. The corresponding gating workflow for JC-1 and H 2 DCFDA analysis is shown in (G, H) Representative transmission electron micrographs showing mitochondrial ultrastructure in 4T1 control (gNC) and Gpat4 -deficient (g Gpat4 ) cells. Red arrows indicate mitochondria. White boxes in (G) denote the regions enlarged in (H). Compared with control cells, Gpat4 -deficient cells display evident mitochondrial abnormalities, including altered shape and disorganization of cristae. Scale bars: 1 μm (G) and 0.5 μm (H). (I) Quantification of cytosolic mtDNA amplicons shown in the panel, including mtCytb (1), mtCytb (2), mtDloop1, mtDloop2, mtDloop3 and mtND6, together with nuclear DNA Tert as a contamination control, in cytosolic fractions prepared from control and Gpat4 -depleted cells. Bars denote mean ± s.e.m., P values are indicated in the respective panels, and exact P values are shown where provided and otherwise significance is denoted as ns or by asterisks. The statistical methods are described in the Materials and methods section. ns, not significant; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.000 1.

Article Snippet: To assess mitochondrial stress, intracellular reactive oxygen species were measured using H 2 DCFDA (Solarbio), and mitochondrial membrane potential was measured using JC-1 (MCE) by flow cytometry.

Techniques: Activation Assay, Comparison, Mutagenesis, Plasmid Preparation, Control, Membrane, Transmission Assay

Journal: International Journal of Molecular Medicine

Article Title: Microbiota-derived indole-3-propionic acid reprograms bone marrow stem cell fate via PPARγ suppression to rescue osteoporosis

doi: 10.3892/ijmm.2026.5879

Figure Lengend Snippet: IPA protects mBMSCs against H 2 O 2 -induced apoptotic injury. (A) Effects of IPA on mBMSC viability over 1, 2, and 3 days, as determined by the CCK-8 assay. (B) Effects of IPA pretreatment on mBMSC viability following H 2 O 2 exposure for 12, 24, and 48 h, as determined by the CCK-8 assay. (C) Live/dead staining of mBMSCs on day 1 using propidium iodide (dead cells) and Calcein-AM (live cells). (D) Quantification of the dead-cell ratio based on live/dead staining. (E) Mitochondrial membrane potential of H 2 O 2 -treated mBMSCs with or without IPA treatment for 1 day, assessed by JC-1 staining. (F) Quantification of the red/green fluorescence ratio from JC-1 staining. All experiments were repeated at least three times. Data are presented as the mean ± SD. ** P<0.01 compared with the control group. IPA, indole-3-propionic acid; mBMSCs, mouse bone marrow stromal cells; CCK-8, Cell Counting Kit-8; ns, not significant.

Article Snippet: MMP was assessed using a JC-1 assay kit (MedChemExpress).

Techniques: CCK-8 Assay, Staining, Membrane, Fluorescence, Control, Cell Counting

Journal: Bioactive Materials

Article Title: Inhalable PD-L1-engineered hybrid cellular vesicles suppress excessive neutrophil activation and restore mitochondrial homeostasis to alleviate ischemia–reperfusion lung injury and pneumonia

doi: 10.1016/j.bioactmat.2026.03.024

Figure Lengend Snippet: Res-PD-L1@nmEVs Restores Mitochondrial Homeostasis and Improves Energy Metabolism BEAS-2B cells were pretreated with Res, nEVs, PD-L1@mEVs, PD-L1@nmEVs, or Res-PD-L1@nmEVs followed by H/R stimulation for subsequent analysis. (A) Representative immunofluorescence images showing the expression and localization of PINK1 (green) and the mitochondrial marker TOMM20 (red), indicating activation of mitophagy. Nuclei were stained with DAPI (blue). Scale bar: 50 μm. (B) Quantitative analysis of PINK1 fluorescence intensity. (C) Expression and localization of autophagy-related proteins LC3B and Beclin-1 detected by immunofluorescence. (D-E) Quantitative analysis of LC3B (D) and Beclin-1 (E) fluorescence intensity. (F) Mitochondrial membrane potential assessed by JC-1 staining and flow cytometry. (G) Oxygen consumption rate (OCR) profiles of lung epithelial cells under different treatments. (H-K) Key mitochondrial respiration parameters: basal respiration (H), maximal respiration (I), proton leak (J), and ATP production (K). (L) Representative confocal microscopy images of mitochondria stained with MitoTracker (green) and lysosomes stained with LysoTracker (red), demonstrating mitochondrial-lysosomal colocalization. Scale bar: 5 μm ∗ vs. Control; # vs. H/R; & vs. H/R + PD-L1@nmEVs, p < 0.05.

Article Snippet: Changes in mitochondrial membrane potential were assessed by JC-1 staining (C2003S, Beyotime).

Techniques: Immunofluorescence, Expressing, Marker, Activation Assay, Staining, Fluorescence, Membrane, Flow Cytometry, Confocal Microscopy, Control

Journal: Frontiers in Pharmacology

Article Title: Piroxicam accelerates diabetic foot ulcer healing via ERα-dependent mitochondrial protection and oxidative stress relief

doi: 10.3389/fphar.2026.1834818

Figure Lengend Snippet: Piroxicam alleviated mitochondrial oxidative stress and restored the mitochondrial function of HG-induced HaCaT cells. After 24 h of induction with HG (60 mM), HaCaT cells were treated with piroxicam (0, 1.2, 3.7, 11, 33, 100 nM) for 24 h. (A) The level of ROS within mitochondria was assessed using MitoSOX staining, and red fluorescence indicated mitochondrial superoxide (scale bar = 50 μm). (B) HaCaT cells were stained with JC-1 dye. Red fluorescence (JC-1 aggregates) indicated healthy mitochondria with a high MMP, while green fluorescence (JC-1 monomers) indicated mitochondrial membrane depolarization (scale bar = 50 μm). (C) The expression of mitochondrial respiratory chain complex proteins, including ATP5A1 (complex V), MT-CO2 (complex IV), UQCRC1 (complex III), SDHB (complex II), and NDUFB8 (complex I), of HaCaT cells was determined by Western blotting. (D) Quantitative analysis of protein levels of ATP5A1, MT-CO2, UQCRC1, SDHB, and NDUFB8 was performed. (E) The expression of mitochondrial dynamics-related proteins, including DRP1, MFN1, MFN2, and OPA1, was measured using Western blotting. (F) Quantitative analysis of protein levels of DRP1, MFN1, MFN2, and OPA1 was performed. (G) HaCaT cells were stained with MitoTracker and observed using a confocal microscope (scale bar = 10 μm). All data were presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 compared to the 0 nM group ( n = 3). Abbreviations: HG, high glucose; SD, standard deviation; ROS, reactive oxygen species; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide; MMP, mitochondrial membrane potential; ATP5A1, ATP synthase F1 subunit alpha; MT-CO2, mitochondrial-encoded cytochrome c oxidase 2; UQCRC1, ubiquinol-cytochrome c reductase core protein 1; SDHB, succinate dehydrogenase complex iron-sulfur subunit B; NDUFB8, NADH: ubiquinone oxidoreductase subunit B8; DRP1, dynamin-related protein 1; MFN1, mitofusin 1; MFN2, mitofusin 2; OPA1, optic atrophy 1.

Article Snippet: MMP was detected using a 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) staining probe (MedChemExpress, Monmouth Junction, NJ, United States) following the manufacturer’s instructions.

Techniques: Staining, Fluorescence, Membrane, Expressing, Western Blot, Microscopy, Standard Deviation