Figure 1. Mitochondrial stress alters mitochondrial function. (A) Schematic representation of the mechanism of action of the compounds selected for the analysis. Acti, actinonin; Dox, doxycycline; MB, MitoBloCK-6. (B) Mitochondrial membrane potential after 24 h of treatment with the selected chemicals. Tetramethylrhodamine, methyl ester (TMRM) was used to determine mitochondrial membrane potential (n = 4 independent experiments; mean values ± SEM). (C) Mitochondrial and (D) total ROS levels after 24 h of treatment with the selected chemicals. Dichlorofluorescin diacetate (DCF-DA) reflects total cellular ROS levels, whereas MitoSox measure mitochondrial superoxide level. RFU, relative fluorescence units (n = 4 independent experiments; mean values ± SEM). (E) Oxygen consumption rate (OCR) of cells treated with the different compounds. Dashed vertical lines indicate the subsequent addition of the ATPase inhibitor oligomycin (Olig.), the uncoupling reagent FCCP and the inhibitors of the electron transport chain rotenone/antimycin A (Rot/Ant). (F and G) Boxplots representing OCR (F) in basal conditions and (G) after treatment with the uncoupler FCCP (maximal respiration). (H) Boxplot representing the ATP-dependent respiration (oligomycin-sensitive respiration) calculated as the difference in OCR before and after the addition of oligomycin. (I) Ratio of OCR and extracellular acidification rate (ECAR) as an indicator of the relation between mitochondrial respiration and glycolysis. (J) ECAR in basal conditions as indication of glycolytic rate. For E–J, n = 2 independent experiments, using 10 replicates per experiment; mean values ± SEM of a representative experiment. (K) Inmunoblot analysis showing the effects of the compounds on different mitochondrial OXPHOS subunits (ATPA5, complex V; UCQRC2, complex III; MTCO1, complex IV; SDHB, complex II; and NDUFB8, complex I). HSP90 was used as loading control. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure 1.

Mitochondrial stress alters mitochondrial function. (A) Schematic representation of the mechanism of action of the compounds selected for the analysis. Acti, actinonin; Dox, doxycycline; MB, MitoBloCK-6. (B) Mitochondrial membrane potential after 24 h of treatment with the selected chemicals. Tetramethylrhodamine, methyl ester (TMRM) was used to determine mitochondrial membrane potential (n = 4 independent experiments; mean values ± SEM). (C) Mitochondrial and (D) total ROS levels after 24 h of treatment with the selected chemicals. Dichlorofluorescin diacetate (DCF-DA) reflects total cellular ROS levels, whereas MitoSox measure mitochondrial superoxide level. RFU, relative fluorescence units (n = 4 independent experiments; mean values ± SEM). (E) Oxygen consumption rate (OCR) of cells treated with the different compounds. Dashed vertical lines indicate the subsequent addition of the ATPase inhibitor oligomycin (Olig.), the uncoupling reagent FCCP and the inhibitors of the electron transport chain rotenone/antimycin A (Rot/Ant). (F and G) Boxplots representing OCR (F) in basal conditions and (G) after treatment with the uncoupler FCCP (maximal respiration). (H) Boxplot representing the ATP-dependent respiration (oligomycin-sensitive respiration) calculated as the difference in OCR before and after the addition of oligomycin. (I) Ratio of OCR and extracellular acidification rate (ECAR) as an indicator of the relation between mitochondrial respiration and glycolysis. (J) ECAR in basal conditions as indication of glycolytic rate. For E–J, n = 2 independent experiments, using 10 replicates per experiment; mean values ± SEM of a representative experiment. (K) Inmunoblot analysis showing the effects of the compounds on different mitochondrial OXPHOS subunits (ATPA5, complex V; UCQRC2, complex III; MTCO1, complex IV; SDHB, complex II; and NDUFB8, complex I). HSP90 was used as loading control. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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