Figure 4.

The knockdown of TOM40 expression increases the mitochondrial membrane potential. Epithelial ovarian cancer (EOC) cells that stably express sh-control and sh-TOM40 were seeded at 2.5 × 10⁶ cells per wells and stained with 2 μM tetraehylbenzimidazolylcarbocyanine iodide (JC-1) for 15 min. The JC-1 staining was measured by fluorescence-activated cell sorting (FACS) analysis and fluorescence microscopy imaging. (A) Representative images of FACS analysis of 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) staining: Quantification of the percentage of cells is shown in the top-left and top-right regions of dot plots to count JC-1 aggregation (red fluorescent). The overlay histograms are representative images of three separate experiments (The white area is sh-control cells, and the red area is sh-TOM40 cells). The results are expressed as the mean ± Standard Deviation (S.D.)., n = 3. (B) Representative fluorescent images of JC-1 staining images: The scale bar is 100 µm. EOC cells that stably express sh-control and sh-TOM40 were trypsinized, and 1 × 10⁶ cells were confined into a round-bottom tube. The cells were stained with a mixture of 100 nM MitoTracker Green FM and 25 nM tetramethylrhodamine ethyl ester (TMRE) for 15 min at 37 °C CO₂ incubator (5% CO₂ and 37 °C). The levels of MitoTracker Green FM and TMRE staining were measured using FACS analysis. (C) Representative images of histograms of MitoTracker and TMRE staining: Quantification of the percentage of cells is shown in the blue line region of histogram. The white area is sh-control cells, while the green or red area is sh-TOM40 cells in histogram. The bar graph displays the relative stained cells. The results are expressed as the mean ± S.D., n = 4 (TOV-112D and OVCAR-3); n = 6 (OVCA-429 and RMUG-S). ^# p > 0.05; * p < 0.05; *** p < 0.001; and **** p < 0.0001. (D) Representative fluorescent images of TMRE staining of EOC cell lines that stably express sh-control or sh-TOM40: The Scale bar is 200 μm.