jc 1 staining probe  (Beyotime)

Journal: Cell Death & Disease

Article Title: Splice-switching of the oncogenic BCS1L isoform suppresses ovarian cancer progression by disrupting mitochondrial function

doi: 10.1038/s41419-026-08495-6

Figure Lengend Snippet: A GO enrichment of proteins captured by the FLAG antibody from A2780 cells overexpressing BCS1L-L or BCS1L-S with FLAG tags. B Mitochondrial basal OCR, ATP production, Maximal OCR, Proton leak, non-mitochondrial respiration, and spare capacity (SC) were evaluated in A2780 cells with BCS1L-L or BCS1L-S overexpression for 72 h and control cells using an Agilent Seahorse XFe24. C ATP content per 10 5 A2780 cells transfected with control, BCS1L-L, and BCS1L-S overexpression vectors for 72 h. D MMP was measured by JC-1 staining in A2780 cells with control and BCS1L-L or BCS1L-S overexpression for 72 h treated with 500 μM H 2 O 2 for 4 h. Red fluorescence indicated normal MMP, while green fluorescence indicated abnormal MMP. E Apoptotic cells were analyzed by flow cytometry using Annexin V/7-AAD staining in A2780 cells with control and BCS1L-L or BCS1L-S overexpression for 48 h treated with 500 μM H 2 O 2 for 4 h. Statistical analysis of apoptotic cells included early (Q2) and late (Q3) apoptotic cells. F Immunofluorescence of COX4 (red) in A2780 and HEY cells with BCS1L knockdown and control cells showing mitochondria morphology. DAPI was used to visualize the nuclei. Scale bars, 10 µm. G and H Mitochondrial basal OCR, maximal OCR, and SC (spare capacity) were evaluated in A2780 and HEY cells with BCS1L knockdown for 72 h using an Agilent Seahorse XFe96. I ATP content per 10 5 cells in three ovarian cancer cell lines with BCS1L knockdown for 48 h (three independent experiments). J MMP was measured by JC-1 staining in A2780 and HEY cells with BCS1L knockdown for 72 h. Red fluorescence indicated normal MMP, while green fluorescence indicated abnormal MMP. K Apoptotic cells were analyzed by flow cytometry using Annexin V/7-AAD staining in A2780 and HEY cells with BCS1L knockdown for 72 h. Statistical analysis of apoptotic cells included early (Q3) and late (Q2) apoptotic cells. All results were repeated in three independent experiments and evaluated by a two-tailed unpaired Student’s t -test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Data are shown as the mean ± SD.

Article Snippet: MMP and ROS production were assessed by flow cytometry using the fluorescent probes JC-1 (MultiSciences Biotech,70-MJ101) and DCFH-DA (Beyotime, S0027).

Techniques: Over Expression, Control, Transfection, Staining, Fluorescence, Flow Cytometry, Immunofluorescence, Knockdown, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Splice-switching of the oncogenic BCS1L isoform suppresses ovarian cancer progression by disrupting mitochondrial function

doi: 10.1038/s41419-026-08495-6

Figure Lengend Snippet: A Transmission electron microscopy in control and A2780 cells with USP39 knockdown. Scale bars, 2 µm. Morphometric analysis of mitochondrial cristae was performed in a blinded fashion on at least five mitochondria per cell from six randomly selected cells, and maximal cristae width was measured using Image J. B Silver staining of BN-PAGE indicated mitochondrial OXPHOS assembly in control and USP39 knockdown A2780 cells. C and D Electron transport chain Complex III activities in control and USP39 knockdown A2780 cells were evaluated by substrate-uncoupler-inhibitor-titration (SUIT) on a high-resolution Oxygraph-2k respirometer. Complex activities were normalized to that of CS ( n = 3). Quantification of OCR measurements in A2780 cells ( E ) and HEY cells ( F ) after USP39 knockdown with the pharmacological inhibitors of metabolism (left). Individual parameters of mitochondrial function, including basal respiration, ATP production, proton leak, and spare respiratory capacity, were calculated according to the manufacturer’s protocol with non-mitochondrial oxygen consumption adjustment (right). Rot/AA, rotenone/antimycin A. G Quantification of ATP levels in A2780 cells for investigating the potential of BCS1L to rescue the loss of USP39. H and I ROS distribution was measured by flow cytometry in A2780 cells transfected with siUSP39 and BCS1L overexpression vector for 48 h. J Measurements and quantification of MMP (by JC-1) in A2780 cells with USP39 knockdown and BCS1L overexpression for 72 h. K Apoptotic A2780 cells after USP39 depletion together with BCS1L overexpression for 48 h were analyzed by flow cytometry using Annexin V/7-AAD staining. Three biological replicates were conducted in all functional experiments, and the p -value was calculated by two-tailed Student’s unpaired t -test ( A , C–G , I–K ), * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns: not significant. Data are shown as the mean ± SD.

Article Snippet: MMP and ROS production were assessed by flow cytometry using the fluorescent probes JC-1 (MultiSciences Biotech,70-MJ101) and DCFH-DA (Beyotime, S0027).

Techniques: Transmission Assay, Electron Microscopy, Control, Knockdown, Silver Staining, Titration, Flow Cytometry, Transfection, Over Expression, Plasmid Preparation, Staining, Functional Assay, Two Tailed Test