Measurement of mitochondrial bioenergetics

The parameters of mitochondrial bioenergetics (Fig. 1A) were determined using an Oroboros-2 k oxygraph (Oroboros Instruments, Innsbruck, Austria). Datalab software (Oroboros Instruments) was used for data acquisition and analysis. All measurements were performed at 37 °C in MIRO5 medium (110 mM sucrose, 60 mM K-lactobionate, 0.5 mM EGTA, 1 g/liter BSA essentially fatty acid free, 3 mM MgCl₂, 20 mM taurine, 10 mM KH₂PO₄, and 20 mM HEPES, adjusted to pH 7.1) with continuous stirring at 750 rpm in the presence of mitochondrial substrates and inhibitors, according to the slightly modified procedures that were described previously^39,40.

MCF-7, MDA-MB-231 and HUVEC-ST cells were seeded on Petri dishes (3,000,000 cells/dish) and cultured for 1 day at 37 °C before treatment with two forms of DOX. DOX–Tf as well as free DOX (IC₅₀ concentration) were incubated with the cells for 4, 24 and 72 h at 37 °C. The cells were thoroughly washed with PBS and harvested by trypsin–EDTA (Sigma–Aldrich, St Louis, MO, USA), pelleted, and suspended in respiration buffer (MIR05). The cell suspensions were added to each chamber (Fig. 1B,C) and stabilized under the routine respiration conditions. Digitonin (10 µg/mL) was added to access the mitochondria with different respiratory substrates (the optimal concentration of digitonin was evaluated in an independent set of experiments) (Fig. 1C). Respiration through Complex I was measured by adding glutamate (5 mM) and malate (5 mM), then the maximal respiratory capacity with convergent electron flow through both Complex I and Complex II was achieved by adding succinate (10 mM). Subsequently, ADP addition at saturating concentration (0.25 mM) caused the maximal OXPHOS capacity (or State 3), after which ATP synthase was inhibited by oligomycin (2 µg/mL) to evaluate State 4. In all experiments, the lack of significant increase in respiration after addition of cytochrome c confirmed the integrity of the outer mitochondrial membrane. The maximal electron flow through respiration without OXPHOS (ETS state) was assessed by the stepwise (5–20 µL) titration of FCCP (2.5–10 µM). Uncoupled Complex-I-linked respiration was achieved by adding rotenone (0.5 µM). Finally, the ETS was inhibited by antimycin A (2.5 µM) to obtain the residual oxygen flux (ROX). ROX after antimycin addition was subtracted from the steady-state respiration values. Finally, the respiration values were calculated as the decrease in the oxygen concentration at time measured in closed chambers, and expressed per milligram of protein. The protein concentration in each sample was determined by the bicinchoninic acid assay.