13c d glucose

MDA-MB-231 parental cells or a DTCC subset (generated as described above) were incubated for 8 hours with ¹³C-D-Glucose (Cambridge Isotope Labs). Isolation was performed as described previously(71 (link)). Briefly, cells were washed once with 1 mL cold (4 °C) 0.9% NaCl and 1 mL of extraction mixture was added containing 80% methanol with metabolomics amino acid mix MSK-A2-1.2 (Cambridge Isotope Labs). Isolation of cells and subsequent steps were performed using reagents stored at −80 °C and the collection was done on dry ice to mitigate metabolic flux during isolation. Dried metabolite extracts were resuspended in 100 μL water and subjected to LC/MS analysis on a QExactive mass spectrometer coupled to a Dionex Ultimate 3000 UPLC system. Labeled and unlabeled metabolites were identified by exact mass (10 ppm window) and retention time (0.5 min window) referencing an in-house library of chemical standards. Fractional labeling of each isotopomer was calculated as the raw peak area of the isotopomer divided by the sum of raw peak areas for all isotopomers of that metabolite. Pool sizes were calculated as the sum of raw peak areas of all isotopomers of a metabolite divided by the raw peak area of the appropriate internal standard. The internal standard used for each metabolite was pre-selected based on similarity of chemical structure and retention time.

A modified Eagle’s minimum essential medium (mEMEM) was prepared using the following components (per L): 10 mL 100X non-essential amino acids (Sigma M7145), 20 mL 50X essential amino acids (Sigma 5550), 10 mL 100X vitamin mix (Thermo Fisher 1112052), 10 mL 100X sodium pyruvate (Sigma 5280), 10 mL 100X phenol red sodium salt (Sigma 3532), 292 mg L-glutamine, 200 mg CaCl₂, 97 mg MgSO₄, 400 mg KCl, 1.5 g NaHCO₃, 140 mg NaH₂PO₄·H₂O, and 6.8 g NaCl. ¹²C D-glucose (Sigma) or ¹³C D-glucose (Cambridge Isotope Labs, CLM-1396–5) was added to a final concentration of 1.0g/L or 1.33 g/L, respectively. The medium was then vacuum-filtered through a 0.22-μm filter, followed by the addition of 10% FBS (Thermo), penicillin G (100 U/mL) and streptomycin (100 μg/mL), prior to storage at 4°C.

The Arf binding domain of Hdm2 (residues 210–304) with an N-terminal polyhistidine tag was expressed in Escherichia coli (E. coli) BL21 (λDE3) from the pET28a expression vector (Novagen) as described previously [5 (link)]. ¹³C/¹⁵N-labeled Hdm2-ABD was expressed using MOPS-based minimal media [8 (link)] containing [¹³C] D-glucose and ¹⁵NH₄Cl (Cambridge Isotope Laboratories). Cultured cells were harvested by centrifugation and lysed in 25 mM Tris HCl (pH 8.0), 500 mM NaCl, 5 mM β-mercaptoethanol (BME), and protease inhibitor cocktail (Sigma) by sonication. Lysates were clarified by centrifugation and Urea was added to the clarified extract to a concentration of 3 M. His-tagged Hdm2-ABD was purified by Ni²⁺-NTA affinity chromatography and eluted with buffer containing 6 M urea and 0.5 M Imidazole. Fractions containing Hdm2-ABD were dialyzed against 25 mM Tris HCl (pH 8.0), 150 mM NaCl, 5 mM BME and treated with thrombin to cleave the His tag. Cleaved Hdm2-ABD was buffer exchanged by dialysis into 25 mM sodium phosphate (pH 7.0), 50 mM NaCl and further purified using anion-exchange chromatography (Q Sepharose; Amersham Pharmacia Biotech, Inc.) using a linear gradient of 0.05′1 M NaCl over 0.1 L. NMR experiments were performed at a protein concentration of 1 mM in 25 mM Sodium Phosphate pH 6.0, 10 mM NaCl, 0.03 % NaN₃, and 10% D₂O.