membrane potential assay kit  (Beyotime)

Journal: Genes & Diseases

Article Title: PDK1 elevation was induced by epigenetic modifications of KDM3A and METTL16 to mediate TKI resistance and cancer development

doi: 10.1016/j.gendis.2025.101947

Figure Lengend Snippet: PDK1 inhibitor JX06 and gefitinib synergistically induced cell apoptosis in gefitinib-resistant lung cancer cells. (A) The protein expression levels of PDK were reduced upon the treatment of PDK1 inhibitor JX06 in PC-9 and PC-9/G cells. (B) B2B and PC-9/G cells were treated with different concentrations of JX06 for 48 h. The cell viabilities were determined by CCK-8. (C – E) The synergy effect between JX06 and gefitinib was determined and analyzed with CompuSyn software. (F) The apoptosis rates were analyzed with flow cytometry after the combined treatment of gefitinib and JX06. (G) The TUNEL assay was performed with the indicated treatment in PC-9/G cells. (H) The cells treated as described were stained with the JC-1 probe and detected using a fluorescence microscope. Red fluorescence indicates the aggregation form of JC-1, showing increased mitochondrial membrane potential (ΔΨm). Green fluorescence indicates the monomeric form of JC-1, which indicates reduced mitochondrial membrane potential (ΔΨm). Data were statistically analyzed with Student’s t -test, and values were shown as mean ± standard deviation. ∗ P < 0.05 and ∗∗ P < 0.01.

Article Snippet: After different treatments for 48 h, the mitochondrial membrane potential of cells was detected with the enhanced mitochondrial membrane potential assay kit using JC-1 probe (Beyotime, Jiangsu, China), and the fluorescence was analyzed with a fluorescence microscope.

Techniques: Expressing, CCK-8 Assay, Software, Flow Cytometry, TUNEL Assay, Staining, Fluorescence, Microscopy, Membrane, Standard Deviation

Journal: iScience

Article Title: CGF induces ROS-mediated metabolic reprogramming and mitochondrial dysfunction to suppress colorectal cancer progression

doi: 10.1016/j.isci.2026.115273

Figure Lengend Snippet: CGF’s effect on ABC transporter pathway, mitochondrial function, and ROS in CRC (A) Flow cytometry analysis of the effect of CGF on ROS levels in HCT116 and HT29 cells. Cells were stained with DCFH-DA, a ROS probe, and fluorescence intensity was measured. The lower panel shows the relative percentage of ROS levels in HCT116 and HT29 cells under different treatments. (B) TEM observation of mitochondrial morphology in HCT116 (top) and HT29 (bottom) cells treated with CGF (50 μM, 24h). Arrows indicate normal mitochondrial morphology (scale bar, 20 μM). (C) JC-1 staining was used to assess how CGF treatment affects the mitochondrial membrane potential in HCT116 and HT29 cells. The change in mitochondrial membrane potential is indicated by the red to green fluorescence ratio (scale bar, 20 μM). (D and E) Assessment of SOD (upper) and CAT (lower) enzyme activities in HCT116 and HT29 cells following CGF exposure. (F and G) RT-qPCR was used to analyze the relative expression levels of ABC transporter genes such as ABCA1 , ABCC2 , ABCB5 , and CFTR in HCT116 (F) and HT29 (G) cells exposed to varying concentrations of CGF. (H) Assays for ATP detection demonstrate the impact of CGF on ATP levels within HCT116 and HT29 cells, with four biological replicates. (I) Flow cytometry analysis of the effect of ATP on ROS levels in HCT116 and HT29 cells. The right panel shows the relative percentage of ROS levels in HCT116 and HT29 cells after ATP treatment. (A, D–I) Data presentation is in the form of mean ± SEM. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001.

Article Snippet: Enhanced mitochondrial membrane potential assay kit with JC-1 , Beyotime , Cat #C2003S.

Techniques: Flow Cytometry, Staining, Fluorescence, Membrane, Quantitative RT-PCR, Expressing

Journal: Nucleic Acids Research

Article Title: Development and application of G4-Flame as a visual biosensor for G4-DNA

doi: 10.1093/nar/gkag179

Figure Lengend Snippet: G4-Flame visualizes G4 Structures in cell mitochondria. ( A ) Schematic diagram of Mito-G4-Flame cell-line construction. Green: MTS sequence, yellow: G4-Flame sequence, and blue: 3× flag tag sequence. Arrows of different colors in the plasmid represent the corresponding functional elements shown in the diagram. ( B, C ). ATP levels and mitochondrial membrane potential (JC-1) in 293T cells transiently transfected with Mito-G4-Flame, compared with empty vector controls. No significant differences were observed between the two groups after transfection. ( D – F ) Subcellular fractionation assay validating Mito-G4-Flame mitochondrial targeting. WB probed with: anti-Tomm70 (mitochondrial marker), anti-GAPDH (cytoplasmic marker), anti-Actin (cytoplasmic marker), and anti-Flag (detecting G4-Flame). Results confirm exclusive mitochondrial localization of Mito-G4-Flame in stably transfected (D) HEK293T, (E) PLC, and (F) SNU 449 cells. Fluorescence images ( G, I , and K ) and co-localization analysis ( H, J , and M ) of HEK293T, PLC, and SNU 449 cells expressing Mito-G4-Flame (ex 485/405 nm, em 520 nm) costained with anti-ATP5A1 (mitochondrial marker). Scale bars: 10 µm. Data information: (B, C) are mean ± SD, unpaired t -test; ns, not significant.

Article Snippet: Twenty-four hours after transfection, the mitochondrial membrane potential was measured using the Beyotime Enhanced Mitochondrial Membrane Potential Assay Kit with JC-1.

Techniques: Sequencing, FLAG-tag, Plasmid Preparation, Functional Assay, Membrane, Transfection, Fractionation, Marker, Stable Transfection, Fluorescence, Expressing

Journal: Nucleic Acids Research

Article Title: Development and application of G4-Flame as a visual biosensor for G4-DNA

doi: 10.1093/nar/gkag179

Figure Lengend Snippet: G4-Flame reveals mitochondrial G4-DNA suppresses mtDNA expression. Fluorescence images ( A ) and quantitative analysis ( B ) of SNU449 cells stably expressing Mito-G4-Flame. Cells were treated with 100 µM PDS for 2, 4, 8, and 10 h at 37 °C, showing a continuous decrease in R485/405 ratios over time. Scale bars: 10 µm. ( C ) PCR analysis of mitochondrial-encoded gene expression in SNU449 cells following PDS treatment across the indicated time points. Heatmap representation shows a time-dependent decrease in mRNA levels with increasing duration of PDS treatment. ( D ) WB analysis showing overexpression of mito-DHX36 in SNU449 cells stably expressing the construct. A strong DHX36 signal is observed, indicating robust expression in the mito-DHX36 stable cell line. Fluorescence images ( E ), colocalization analysis ( F ), and quantitative analysis ( G ) of SNU449 cells stably expressing mito-DHX36. DHX36 shows clear colocalization with mitochondria, and overexpression of mito-DHX36 leads to increased fluorescence intensity. Scale bars: 10 µm. Fluorescence images ( H ) and quantitative analysis ( I ) of SNU449 cells stably expressing Mito-G4-Flame with DHX36 overexpression. Overexpression of DHX36 in the stable cell line leads to an increase in R485/405 ratios. Scale bars: 10 µm. ( J ) qPCR analysis of mitochondrial-encoded gene expression in SNU449 cells stably expressing mito-DHX36. Overexpression of mito-DHX36 in the stable cell line led to increased expression of most mitochondrial-encoded genes. Data information: (J) are mean ± SD, (B) one-way ANOVA with Dunnett’s multiple comparisons test, (G and I) unpaired t- test, (J) two-way ANOVA with Dunnett’s multiple comparisons test; ns, not significant; * P < .05, ** P < .01, *** P < .001, **** P < .0001.

Article Snippet: Twenty-four hours after transfection, the mitochondrial membrane potential was measured using the Beyotime Enhanced Mitochondrial Membrane Potential Assay Kit with JC-1.

Techniques: Expressing, Fluorescence, Stable Transfection, Gene Expression, Over Expression, Construct