*2.3. Cell Respirometry*

The oxygen consumption experiments in intact cells were performed using a high-resolution respirometry equipment O2k meter (Oroboros Instruments, Innsbruck, Austria) according to a method previously described [27]. Cells were pretreated with the corresponding schemes of αM, CDDP or co-incubation for 24 h. Subsequently, cells were washed with PBS, harvested with trypsin, and quantified by the trypan blue assay. Determinations were made using 2 mL of culture medium with 10% FBS. Each experiment was initiated by the addition of approximately 2.5 million cells, the respiratory parameters were defined as: 1. Routine respiration, corresponding to oxygen consumption with presence only of cells. 2. Leak, corresponding to cellular oxygen consumption in the presence of 5 μM oligomycin. 3. Maximum capacity of electron transfer system (E) was achieved by titrations with 5 μM DNP. 4. Respiratory control (RC) corresponding to the ratio basal/leak. 5. Respiration associated with oxidative

phosphorylation (P) was calculated by the formula Routine-Leak. All parameters were corrected by subtracting the non-mitochondrial respiration, obtained by the addition of 1 μM rotenone plus 5 μM antimycin A, and normalized by the number of cells.

Complex I (CI)-linked respiration was measured in a different experiment by the rest of routine respiration minus the respiration after inhibition with 1 μM rotenone. The activity of complex IV (CIV) was evaluated in intact cells in culture medium with 10% FBS supplemented with 0.5 μM rotenone plus 2.5 μM antimycin A. Oxygen consumption was stimulated by the addition of 0.5 mM TMPD plus 2 mM ascorbate. CIV activity was corrected by oxygen consumption in the presence of the appropriate inhibitor (100 mM NaN3). The results were normalized by the number of cells.
