Journal: Cell Death and Differentiation

Article Title: Bedaquiline, an FDA-approved drug, inhibits mitochondrial ATP production and metastasis in vivo, by targeting the gamma subunit (ATP5F1C) of the ATP synthase

doi: 10.1038/s41418-021-00788-x

Figure Lengend Snippet: A ATP5F1C is overexpressed in the ATP-high cell subpopulation. After metabolic fractionation, the ATP-high and ATP-low subpopulations were subjected to Western blot analysis with a mono-specific antibody probe that specifically recognizes ATP5F1C. Note that ATP5F1C is overexpressed in the ATP-high subpopulation in all four cell lines tested. B ATP levels after metabolic fractionation. The relative amount of ATP in the ATP-high and ATP-low cell subpopulations was independently validated using Cell-Titer-Glo. 2D monolayer cells were first stained with BioTracker ATP-Red 1 and sorted by ATP content, using a flow cytometer. After cell counting, equal numbers of single cells were then used to evaluate their relative luminescence content. In this series of experiments, we used a cut-off of 10% to define the ATP-high and ATP-low cell populations. Note that this metabolic fractionation scheme can be successfully applied to other breast cancer cell lines. Data represent the mean fold increase over ATP-low 10% cells ±SD, n = 3. Unpaired t test, * p < 0.05, ** p < 0.005, *** p < 0.0005. C Mammosphere formation. Anchorage-independent growth was quantitated using the 3D mammosphere formation assay. In this series of experiments, we used a cut-off of 10% to define the ATP-high and ATP-low cell populations. Cells in 2D monolayers were first stained with BioTracker ATP-Red 1 and sorted by ATP content, using a flow cytometer. After cell counting, 5 × 10 3 cells were seeded onto a poly-HEMA coated 6-well plate and counted after 5 days. Note that the ATP-high cell population of MCF7, T47D, MDA-MB-231, and MDA-MB-468 cells all showed an increased capacity for 3D anchorage-independent growth. Data represent the mean fold increase over ATP-low 10% cells ±SD, n = 3. Unpaired t test, *** p < 0.0005, **** p < 0.0001. D ATP levels remain high, even 24 h after plating. ATP levels in different MCF7 cell subpopulations were quantitated using Cell-Titer-Glo analysis. After cell counting, equal numbers of single cells were then used to evaluate their relative ATP content by luminescence using the Varioskan ™ LUX plate reader, 24 h after plating. For these experiments, which required larger numbers of cells, we used a cut-off of 10% to define the ATP-high and ATP-low cell populations. Note that ATP-high cells showed a greater than threefold increase in ATP levels, as compared to ATP-low cells. Data represent the mean fold increase ±SD over ATP-low 10% cells, n = 3. Unpaired t test, **** p < 0.0001. E Metabolic flux analysis of ATP-high MCF7 cells. The OCR (oxygen consumption rate) and the ECAR (extracellular acidification rate) were determined using the Seahorse XFe96, via metabolic flux analysis. Note that the ATP-high MCF7 cell population shows an increase in both basal and maximal respiration, as well as mitochondrial ATP-production. The ATP-high MCF7 cell population also shows an increase in glycolysis. Cell populations were analyzed 24 h after plating. Data represent the % average fold increase ±SD over ATP-low 10% cells, n = 3. Unpaired t test, ns = not significant, * p < 0.05, ** p < 0.005, *** p < 0.0005 . F Metabolic flux analysis of ATP-high MDA-MB-231 cells. The OCR (oxygen consumption rate) and the ECAR (extracellular acidification rate) were determined using the Seahorse XFe96, via metabolic flux analysis. Note that the ATP-high MDA-MB-231 cell population shows an increase in both basal and maximal respiration, as well as mitochondrial ATP-production. The ATP-high MDA-MB-231 cell population also shows an increase in glycolytic reserve and glycolytic reserve capacity. Cell populations were analyzed 24 h after plating. Data represent the % average fold increase ±SD over ATP-low 10% cells, n = 3. Unpaired t test, ns = not significant, * p < 0.05, ** p < 0.005, *** p < 0.0005. G Cell cycle progression. Note that the ATP-high cell subpopulation is hyper-proliferative, with increases in cell cycle progression, showing a strong shift away from the G0/G1-phase, toward the S-phase and the G2/M-phase. Quantitatively similar results were obtained with MCF7, T47D, MDA-MB-231, and MDA-MB-468 cell lines. In this series of experiments, we used a cut-off of 10% to define the ATP-high and ATP-low cell populations. The percentage of cells in the G0/G1, S, and G2/M phases of the cell cycle are reported. The results are expressed as the mean from three independent experiments, Unpaired t test, ** p < 0.005, *** p < 0.0005, **** p < 0.0001. H Tempo-ATP: fluorescent-protein biosensor. Tempo-ATP is a fluorescent-protein biosensor for the detection of cytosolic ATP levels. This protein biosensor was stably expressed in MCF7 cells. The Tempo-ATP-biosensor allows for the measurement of ATP levels in a real-time fashion. As such, high GFP-fluorescent content is a reporter for high ATP content within the cells. The protein sequence of the ATP-binding portion of the biosensor is shown in yellow lettering, while the while GFP-like protein sequence is shown in green. Cells were sorted by GFP content, using flow cytometer (excitation = 517–519 nm; emission = 535 nm). I Tempo-ATP: mammosphere formation. Anchorage-independent growth was quantitated, using the 3D mammosphere formation assay. In this series of experiments, we used a cut-off of 10% to define the GFP-high and GFP-low cell populations. Cells in the monolayer were first sorted for GFP content, by flow cytometry. After cell counting, 5 × 10 3 cells were seeded in poly-HEMA coated 6-well plates and counted after 5 days. Note that the number of 3D mammospheres formed was nearly fourfold increased in GFP-high MCF7 cells, as compared with GFP-low MCF7 cells, respectively. Data represent the mean fold increase over GFP-low 10% cells ±SD, n = 3. Unpaired t test, **** p < 0.0001. J Tempo-ATP: metabolic analysis. Metabolic profiling of key metabolites in GFP-high and GFP-low TempoATP-MCF7 cells. Luminescence levels were measured in GFP-high and GFP-low TempoATP-MCF7 cell subpopulations, using Promega kits (Cell-Titer-Glo, GSH/GSSG-Glo, NADP-NADPH-Glo, and NAD-NADH-Glo). Cells were sorted by GFP content, using flow cytometry. After cell counting, equal numbers of single cells were then used to evaluate their relative luminescence content. Note that GFP-high MCF7 cells showed a near 2-fold increase in ATP levels; a 2-fold increase in reduced glutathione levels; a ~1.5-fold increase in NADP-NADPH levels; and >2-fold increase in NAD-NADH levels, all relative to GFP-low cells. Data represent the mean fold increase over GFP-low 10% cells ±SD, n = 4. Unpaired t test, ** p < 0.005, *** p < 0.0005, **** p < 0.0001. K Tempo-ATP: cell cycle progression. The GFP-high MCF7 cell subpopulation is hyper-proliferative, with increases in cell cycle progression, showing a strong shift away from the G0/G1-phase, towards the S-phase and the G2/M-phase. In this series of experiments, we used a cut-off of 10% to define the GFP-high and GFP-low cell populations. The percentage of cells in the G0/G1, S, and G2/M phases of the cell cycle are reported. The results are expressed as the mean from three independent experiments, Unpaired t test, *** p < 0.0005, **** p < 0.0001.

Article Snippet: Reagents for profiling the intracellular levels of metabolites (ATP, GSH, NADH and NADPH) after cell lysis, were all obtained from Promega, Inc. ER(+) [MCF7 and T47D] and triple-negative [MDA-MB-231 and MDA-MB-468] human breast cancer cell lines were obtained from the American Type Culture Collection (ATCC).

Techniques: Fractionation, Western Blot, Staining, Flow Cytometry, Cell Counting, Tube Formation Assay, Stable Transfection, Sequencing, Binding Assay

Journal: The Journals of Gerontology Series A: Biological Sciences and Medical Sciences

Article Title: Metabolites Associated With Lean Mass and Adiposity in Older Black Men

doi: 10.1093/gerona/glw245

Figure Lengend Snippet: Each point represents the absolute Pearson partial correlation between a body composition measure and a metabolite in the Health ABC Study sample. Triangles (red online) represent alkaloids, circles (yellow online) are benzenoids, squares (green online) are lipids, diamonds (blue online) are nucleosides, short dashes (navy online) are organic acids, crosses (purple online) are organoheterocyclics, long dashes (magenta online) are unclassified. Correlations are adjusted for study site, age and smoking.

Article Snippet: Metabolite Profiling A total of 350 metabolites were measured using the metabolite profiling platform of the Broad Institute of the Massachusetts Institute of Technology.

Techniques: