U2OS_p53_Carbon_Tracing

We used mass spectrometry-based metabolite profiling to identify differential utilization of metabolic pathways between U2OS and U2OSp53KO cells. Labeled glucose was used to monitor pathway activity, both at steady state and following exposure to etoposide.  

Isotope tracing experiments were performed using heavy D-13C6-Glucose. Cells were seeded on 6-well plates at 200,000 cells per well. After adherence overnight, cells were treated with DMSO or 31.6 µM etoposide. Eight hours prior to the collection of each timepoint, the growth medium was swapped with glucose-free DMEM supplemented with 10 mM D-13C6-Glucose. At the indicated timepoints, samples were collected in parallel for metabolite extraction or protein quantification. For total protein quantification, cells were trypsinized and pelleted. Pellets were lysed using SDS lysis buffer and quantified using a BCA assay. For extraction of metabolites, growth media was removed, and cells were washed 2 times with ice-cold PBS. With the 6-well plate on dry ice, cells were submerged in 500 µL 80% MeOH. Samples were then incubated at -80°C for 15 minutes. Cell scrapers were used to harvest each sample, and sample wells were washed with an additional 300 µL of 80% MeOH. Samples were vortexed at 4°C for 10 minutes and then centrifuged at top speed for 10 minutes at 4°C. Supernatant was transferred to a new tube and samples were dried using a speed vac. Dried pellets were resuspended in 100 µL of water and vortexed for 10 minutes at 4°C. Samples were then spun for 10 minutes at top speed at 4°C, andsupernatant from each sample was transferred to an LC-MS vial. A QExactive Plus Quadrupole Orbitrap Mass Spectrometer equipped with a HESI II probe (Thermo Fisher Scientific) was then used to perform Mass Spectrometry. Metabolites were quantified by integrating peaks in TraceFinder 5.1 (Thermo Fisher Scientific). Mass tolerance was set to 5 ppm and expected retention times were benchmarked using an in-house library of chemical standards. Natural abundance of heavy isotopes was corrected using IsoCorrectoR (Bioconductor), and each sample was normalized to the amount of total protein. Fold-change and significance was determined using a custom MATLAB script.