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: G3: Genes|Genomes|Genetics

Article Title: Escherichia coli Metabolite Profiling Leads to the Development of an RNA Interference Strain for Caenorhabditis elegans

doi: 10.1534/g3.119.400741

Figure Lengend Snippet: Metabolomic profiles of three E. coli strains and their distinct effects on C. elegans fat storage and mitochondrial dynamics. A) Fat content levels of day-2-old C. elegans adults are quantified using SRS microscopy imaging. When compared to those grown on OP50, worms grown on HB101 and MG1655 show decreased fat storage in the intestine. Example SRS images are presented from three independent trials and intestinal areas for quantification are outlined. B) Mitochondrial morphology is examined in day-3-old C. elegans adults carrying a mitochondria-localized-GFP reporter in the intestine, and classified into three categories for quantification: filamented, fragmented, or intermediate. When compared to those grown on OP50, worms grown on HB101 and MG1655 show decreased mitochondrial fragmentation in the intestine. Phenotypes were noted in a double-blind fashion from three independent trials. C) PCA analysis of E. coli metabolomics profiles shows clustering between OP50 and HB101 that is separated from MG1655. D) Clustering of different E. coli samples based upon metabolite levels shows that HB101 and OP50 have a similar pattern distinct to MG1655. E) Heat maps show relative fold changes in a log scale of significantly altered metabolites in MG1655 and HB101 when compared to OP50. Five independent metabolomics profiles for each strain are presented. Statistical significance indicated by asterisk, * P = <0.05, ** P = <0.005. Error bars represent SEM. Statistical analysis performed using Welch’s two-sample two-sided t -test (E), Student’s t -test (A), or Chi-Squared test (B).

Article Snippet: E. coli metabolite profiling was performed at Metabolon, Inc (Durham, NC) with 5 biological replicates of each test bacterial strain (MG1655, OP50, HB101).

Techniques: Microscopy, Imaging

Journal: G3: Genes|Genomes|Genetics

Article Title: Escherichia coli Metabolite Profiling Leads to the Development of an RNA Interference Strain for Caenorhabditis elegans

doi: 10.1534/g3.119.400741

Figure Lengend Snippet: Bacterial metabolite Betaine regulates fat storage in the host. A) Simplified one-carbon methyl cycle in E. coli , highlighting the methyl donation interactions between four key metabolites, methionine, homocysteine, dimethylglycine, and betaine. Arrows indicate alterations in MG1655 (green) and HB101 (purple) when compared to OP50. B) Quantitative comparison of one-carbon methyl cycle metabolite levels show that betaine levels are increased over threefold and twofold in MG1655 and HB101, relative to OP50, respectively, and the methionine level in HB101 is decreased compared to that in OP50. C) Supplementation of betaine but not glucose suppresses the fat storage increase in C. elegans grown on OP50. Graphical representation of relative whole intestine fat content levels as measured by SRS microscopy from three independent trials. D) Neither betaine nor glucose supplementation affects morphological difference of mitochondria in C. elegans grown on HB101, MG1655, and OP50. Graphical representation of mitochondrial dynamics in the intestinal cells from double-blind analyses of three independent trials. Statistical significance indicated by asterisk, * P = <0.05, ** P = <0.005. Error bars represent SEM. Statistical analysis performed using Student’s t -test (B & C), or Chi-Squared test (D).

Article Snippet: E. coli metabolite profiling was performed at Metabolon, Inc (Durham, NC) with 5 biological replicates of each test bacterial strain (MG1655, OP50, HB101).

Techniques: Comparison, Microscopy

Journal: Scientific Reports

Article Title: Exposure of Methicillin-Resistant Staphylococcus aureus to Low Levels of the Antibacterial THAM-3ΦG Generates a Small Colony Drug-Resistant Phenotype

doi: 10.1038/s41598-018-28283-3

Figure Lengend Snippet: ( a ) Characteristic 1D 1 H NMR spectrum of the protein- and lipid-free intracellular metabolite extract of wild-type S. aureus recorded at 600 MHz ( 1 H Larmor frequency) magnetic field strength with expanded regions of the spectrum displayed in panels ( b – e ). The NMR signals assigned to several metabolites are indicated. The insert (panel f) displaying the spectral patterns spanning the chemical shift range 2.6 to 2.85 ppm highlights how well the spectral features of aspartate can be fitted to its characteristic spectrum annotated in the Chenomx TM database (fit is in red) and compared to the experimental spectrum (shown in black). All NMR chemical shifts of the metabolite resonances are referenced to the DSS (sodium salt of 4,4-dimethyl-4-silapentane-1-sulfonic acid) signal set at 0 ppm.

Article Snippet: The Chenomx TM metabolite profiling approach enabled identification of 30 metabolites and deconvoluted the contributions of different metabolites to potentially overlapping signals .

Techniques:

Journal: Scientific Reports

Article Title: Exposure of Methicillin-Resistant Staphylococcus aureus to Low Levels of the Antibacterial THAM-3ΦG Generates a Small Colony Drug-Resistant Phenotype

doi: 10.1038/s41598-018-28283-3

Figure Lengend Snippet: Multivariate Statistical Analysis of WT and SCV Isolates Treated and Untreated with THAM-3ΦG. Intracellular metabolites were collected for WT and SCV isolates treated (T) or untreated with 2 mg/L THAM-3ΦG for 1 hour. ( A ) 2D-PCA plot demonstrating sample separations based on distinct metabolic profiles and with clustering patterns highlighted with 95% confidence intervals as shading; PC1 and PC2 accounted for 76.8% of the variance. The 2D-PCA plot revealed three significantly distinct metabolic profiles: WT (blue), WT-T (cyan), and SC/SC-T (red/green). ( B ) Hierarchal clustering supports the separation of these groups based on Euclidean distance measurements and Ward based clustering analysis of metabolite levels. The data are representative of three biological replicates with two technical replicates of each treatment type.

Article Snippet: The Chenomx TM metabolite profiling approach enabled identification of 30 metabolites and deconvoluted the contributions of different metabolites to potentially overlapping signals .

Techniques:

Journal: Scientific Reports

Article Title: Exposure of Methicillin-Resistant Staphylococcus aureus to Low Levels of the Antibacterial THAM-3ΦG Generates a Small Colony Drug-Resistant Phenotype

doi: 10.1038/s41598-018-28283-3

Figure Lengend Snippet: Two-Dimensional Principal Component Analysis (2D PCA) of the Intracellular Metabolic Profiles of WT and SCV Isolates Grown to Mid-Log Phase. WT (green) and SC (red), each at mid-log growth phase, exhibited distinct metabolite profiles, with principal component −1 (PC-1) accounting for 81.3% of the variance. A total of six biological replicate samples was collected for each WT and SC cell isolate.

Article Snippet: The Chenomx TM metabolite profiling approach enabled identification of 30 metabolites and deconvoluted the contributions of different metabolites to potentially overlapping signals .

Techniques: