Cell Transfection
MCF-7 cells were transfected with small interfering RNA (siRNA) targeting the ATM, LKB1, and AMPKa or a negative control siRNA using Pipette-type electroporator (MicroPorator MP-100, Digital Bio Technology Co., Ltd., Seoul, Korea) as described by the manufacturer’s instructions.Figure 6. Effects of KU-55933 and metformin on TCA metabolites. (A) TCA metabolites were measured by NMR. P values for changes in TCA metabolites are shown in Table S1 (B) Interpretation of metabolic changes observed. [a] ATM is hypothesized to have a role in oxidative phosphorylation, effecting respiratory complex II. Therefore, the ATM inhibitor KU-55933 leads not only to reduced ATP production, but also to accumulation of succinate. [b] KU-55933 also may reduce oxidative phosphorylation by a mechanism involving SCO2, as discussed in the text. [c] Metformin acts to inhibit oxidative phosphorylation, but prior evidence together with our findings of decreased NAD+ suggest a site of action involving respiratory complex I. [d] Both KU-55933 and metformin exposure lead to increased glucose uptake and lactate production, consistent with a compensatory increase in glycolysis following decreased oxidative phosphorylation. [e] Our observations provide evidence for reduced concentrations of TCA cycle intermediates with exposure to either KU-55933 or metformin, but we postulate different reasons for this: metformin may reduce TCA cycle activity because of a reduction in supply of complex I-generated NAD+, while KU-55933 may act to inhibit conversion of succinate to fumarate. ATM, Ataxia Telangiectasia Mutated protein; SCO2, Synthesis of Cytochrome C Oxidase 2; AMPK, AMP-activated protein kinase; TSC1/TSC2, Tuberous Sclerosis 1/Tuberous Sclerosis 2; mTOR, Mammalian Target of Rapamycin complex 1; rpS6, ribosomal protein S6. respiration was assessed in the absence of inhibitors; while proton leak was measured using the ATP synthase inhibitor oligomycin (2.5 mg/1 6 106 cells) and non-mitochondrial respiration using myxothiazol (12.2 mg/1 6 106 cells).
Cell Growth for NMR
MCF-7 Cells were plated at 16106 per petri in 10 replicates and incubated in medium containing 10% FBS. After 24 hrs, the complete medium was replaced with test medium containing vehicle control or metformin or KU-55933 at 37uC. Three MCF7 plates were not extracted but used for cell counting and protein analysis. The average cell counts were 3.37 million per plate for the control cells, 1.9 million per plate for the KU-55933 treated cells and 2.1 million per plate for the metformin treated cells. These counts were used to normalize the NMR and GC/MS metabolite quantitation.
Figure 7. Subcellular localization of ATM. Total MCF-7 cell lysate and MCF-7 cells fractionated into cytoplasmic, nuclear and mitochondrial extracts were immunoblotted with ATM antibody, a-Tubulin (cytoplasmic marker), Ki67 (nuclear marker) and VDAC (mitochondrial marker). The results indicate ATM immunoreactivity in mitochondrial extracts that are negative for cytoplasmic and nuclear markers. NMR Sample Preparation
MCF-7 cells were extracted as described previously [52]. Briefly, tissue culture plates were removed from the incubator and 2 mL of the spent media was collected and placed in an eppendorf tube. The remaining media was aspirated off the plate to waste. The plated cells were washed three times with ice cold isotonic saline solution. Volumes of 500 mL 80% methanol (prechilled to 220uC) were added to the plates on ice. The cells were scraped off the plates and deposited in eppendorf tubes. The plates were rinsed with second 500 mL aliquots of cold 80% methanol and added to the cells. The cells were then lysed by 5 min of sonication, 30 sec on 30 sec off, on ice using Bioruptor UCD-200TM-EX Sonicator (Diagenode, Denville, NJ, USA). The homogenates were then spun down in a 4uC micro centrifuge for 10 min at 13,000 g and the supernatants were removed to new eppendorf tubes. The extracts were dried in a pre-cooled vacuum centrifuge (Labconco Corp. Kansas City, MO, USA) operating at 24uC and stored at 280uC until the day of NMR analysis. For NMR analysis, cell extracts were re-suspended in 220 mL 2 H2O containing 0.2 mM DSS (4,4-dimethyl-4-silapentane-1sulfonic acid), the chemical shift and concentration standard 0.1 mM DFTMP (difluorotrimethylsilanylphosphonic acid) an internal pH standard [53] and 0.01 mM sodium azide. The pH of each sample was manually adjusted to an uncorrected pH of 6.8+/20.1 with HCl or KOH as needed. Medium samples were ultra-filtered using pre-rinsed 3 kDa cut off filters (Nanosep ultra filter, Pall Corp. Port Washington, NY USA). A volume of 195 mL was removed to a separate tube containing 22 mL of a 2 mM DSS and 1 mM DFTMP solution in 2 H2O. The pH of each medium sample was manually adjusted to a pH of 6.8+/20.1 as before. The samples were then transferred to 3 mm NMR tubes (Wilmad, Buena, NJ, USA) for analysis.incubated for 24 hrs with various concentrations of siRNA prior to KU-55933 or metformin treatment.
Protein Extraction and Western Blot Analysis
Cells were washed three times with ice-cold phosphate-buffered saline (PBS) and lysed in 100?00 ml lysis buffer (20 mM Tris HCl (pH 7.5)), 150 mM NaCL, 2.5 mM sodium pyrophosphate, 1 mM ?glycerol phosphate, 1 mM Na3VO4, 1 mM EGTA, 1% Triton, and Complete Protease Inhibitor Cocktail Tablet from Roche Diagnostic (Laval, QC, Canada). Cell debris was removed by centrifugation at 14,000X rpm for 20 min at 4uC. Following the assay for total protein (Bio-Rad, Mississauga, ON, Canada), clarified protein lysates from each experimental condition (40?50 mg) were boiled for 5 min and subjected to electrophoresis in denaturing 8% SDS-polyacrylamide gel for ATM, 12% for SCO2, or 10% SDS-PAGE for other proteins. Separated proteins were transferred to a nitrocellulose membrane and after blocking, the membranes were probed with antibodies of interest. In some cases, developed blots were stripped in stripping buffer (62 mM Tris HCL (pH 6.8), 100 mM ?mercaptoethanol, 2% SDS) to confirm equal protein loading. Horseradish peroxidase-conjugated antirabbit IgG and anti-mouse IgG were used as secondary antibodies. The position of proteins was visualized using the enhanced chemiluminescene reagent ECL.
Mitochondrial Extraction
Mitochondrial isolation was achieved by using the mitochondrial isolation protocol [51]. Mitochondrial pellets were lysed and protein concentration was determined with the PierceH BCA Protein Assay Kit (Thermo Scientific), with bovine serum albumin (BSA) as a standard.
