Fig. 5. Inhibition of H₂O₂-induced mitochondrial dysfunction by I6CA in V79-4 cells. The cells were treated with 300 μM I6CA for 1 h and then exposed to 1 mM H₂O₂ for 24 h. (A) The cells were collected and stained with JC-1. The JC-1 fluorescence intensity was detected to evaluate the changes in the MMP using a flow cytometer. (B) The percentages of cells with JC-1 monomers and aggregates are indicated by bars, and the data represent the mean ± SD of triplicate determinations (***p<0.001 compared with the control group; ###p<0.001 compared with the H₂O₂-treated group). (C) JC-1 fluorescence images of the cells treated with 1 mM H₂O₂ in the presence or absence of 300 μM I6CA are shown. Red fluorescence indicates high membrane potential, and green fluorescence represents low membrane potential. (D) Western blot analysis of cytochrome c levels in the mitochondrial and cytosolic fractions isolated from the cells cultured under the same conditions. Cytochrome oxidase subunit VI (COX IV) and actin serve as protein loading controls for the mitochondria and cytosol, respectively. (E) Whole cell lysates were prepared, and Bax, Bcl-2 and PARP expression was identified by Western blot analysis. The equivalent loading of proteins in each well was confirmed by actin.