Fig 1.
Modulation of astrocytic lactate metabolism by mitochondrial toxicants.
A) Primary culture of cortical astrocytes from mice imaged at 440 nm excitation/535 nm emission. B) Cytosolic lactate accumulation induced by treatment with 32 μM rotenone, 16 μM of antimycin, 5 mM azide and 80 μM of oligomycin. Each data point is from an average of ten cells from three independent experiments. C) Schematic representation of molecular targets for each toxicant used in the primary culture of astrocytes.
Fig 2.
Selection of an oxidative cell line.
A) A panel of epithelial, tumoral cellular lines, and primary astrocytes expressing cytosolic Laconic. Images were taken by confocal microscopy. Scale barr 10 μm. B) Standard protocol for WI determination. Oxidative and glycolytic metabolism were explored in a primary culture of astrocytes by ETC inhibition with 5 mM Azide and stopping lactate transport with 250 μM, respectively. C) Response distribution of pCMBS slope (glycolysis) and azide (OXPHOS). Oxidative cells are in the right lower quadrant. D) WI calculations for each cell using the quotient from pCMBS/azide slopes. Data from three independent experiments.
Fig 3.
Characterization of “MitoTox reporter” cell line.
A) Confocal images from FACS selected clonal MDA-MB-231 cells expressing Laconic. Size bars for 20x and 60x represent 100 μm and 10 μm, respectively. Cytosolic lactate accumulation induced by treatment with: B) 32 μM rotenone, C) 16 μM of antimycin, D) 80 μM of oligomycin, and 10 μM myxothiazol. Each trace is the average of ten cells from one representative experiment.
Fig 4.
Single-well detection of lactate accumulation induced by mitochondrial toxicants.
Mitochondrial dysfunction induced by mitochondrial toxicants was detected in MitoTox Reporter cells in 96 well plates. Measurements using a standard multiplate reader were performed at 5, 10, 30, and 60 minutes. A) 10 mM Lactate, B) 32 μM rotenone, C) 16 μM antimycin, D) 5 mM azide, E) 80 μM oligomycin and F) 10 μM myxothiazol. Solvent control (gray circles): KRH buffer for azide, 0.8% DMSO for rotenone, oligomycin, and myxothiazol and 0.16% ethanol for antimycin. All the experiments were performed at 37°C.
Fig 5.
Enhancement of lactate accumulation by transport-stop protocol.
All the experiments were performed adding 50 μM of pCMBS together with the mitochondrial toxicant. Measurements were performed at 5, 10, 30, and 60 minutes. A) pCMBS control, B) 32 μM rotenone, C) 16 μM antimycin, D) 5 mM azide, E) 80 μM oligomycin and F) 10 μM myxothiazol. Solvent control (gray circles): KRH buffer for azide, 0.8% DMSO for rotenone, oligomycin, and myxothiazol and 0.16% ethanol for antimycin. All the experiments were performed at 37°C.
Fig 6.
High throughput suitability characterization.
The Z’-factor was calculated using wells treated with 32 μM rotenone, 16 μM antimycin, 5 mM azide, 80 μM oligomycin, and 10 μM myxothiazol as positive controls. Negative control wells were treated with 50 μM of pCMBS (gray circles) and nanosensor response control with 10 mM lactate (yellow circles). All the Z’-Factors were calculated at 10 minutes post-treatment. Experiments from three independent assays for each mitochondrial toxicant, performed on different experimental days.
Fig 7.
Inter and intra-well variability analysis.
CV% values were calculated using the average wells from three independent plates.
Fig 8.
IC50 for classical mitochondrial toxicants.
Dose-response Lactate accumulation induced by classical mitochondrial toxicants was measured at: A) 10xE10-5, 10xE10-8, 10xE10-11, 10xE10-14, 10xE10-17 and 10xE10-20 in molar of rotenone and B) antimycin C) 10, 5, 2.5, 1, 0.5, 0.25, 0.1, 0.05, 0.01 and 0.005 in mM of azide D) 16, 10, 8, oligomycin and E) 10, 2, 1, 0.2, 0.1, 0.02, 0.01, 0.002, 0.001, 0.0001 and 0.00001 in μM of myxothiazol. Data was collected at 10 minutes after toxicant treatment at 37°C. Data is the average from three independent experiments.
Table 1.
IC50 Comparison of state-of-art methods to evaluate mitochondrial dysfunction.
Benchmarking analysis comparing the IC50 archived with the Laconic based method and current technology to assess mitochondrial dysfunction.
Fig 9.
A) Schematic representation of the 96 well plate format with their corresponding ΔR% values in pseudo color codification. Red and blue colors represent high and low ΔR%, respectively. Drugs were used at 1, 5 and 10 μM and effects were measured after 30 minutes of treatment at 37°C B) Barr plot of the effect on lactate levels of a panel of drugs at 1, 5, and 10 μM at 37°C after of 30 minutes of pharmacological treatment. Data represent the average of 4 replicate wells of one representative experiment.
Fig 10.
Stability of lactate accumulation.
Lactate accumulation at 5, 10, 30, and 60-minutes of treatment with a panel of drugs at 37 C. Data obtained from treatment with 10 μM of each compound. Data represent the average of 4 replicate wells of one representative experiment.