The detection of changes in the inner electrochemical Δψm in living cells was performed as described previously (35 (link)), using the cationic, lipophilic JC-1 dye (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Carbonyl cyanide 3-chlorophenylhydrazone (CCCP; Sigma-Aldrich; Merck KGaA), a mitochondrial potential disrupter, was used as a control. The melanoma A375 cells were pre-treated with secosteroids at a concentration of 100 nM and then exposed to 2.4 and 12 µM cisplatin or 2.0 and 10 µM dacarbazine for an additional 3 h, or to 75 nM hydrogen peroxide for 1-3 h. Following the treatment with the selected compounds, the cells were harvested and suspended in 1 ml PBS at room temperature. CCCP solution in dimethylsulfoxide (DMSO) was added to the positive control tube only (2 µM final concentration) and the cells incubated at 37°C for 5 min. JC-1 solution (2 µM in DMSO) was added to all tubes and the cells were incubated at 37°C for 15 min, then centrifuged at 1,000 × g for 10 min at room temperature, and resuspended in 500 µl PBS. The samples were kept on ice until they were analyzed on the FACSCalibur flow cytometer using the CellQuest Pro analysis software.