Pyruvate-1-13C pulse-chase analysis by NMR

Three different conditions were tested in hepatocytes: control cells, p53-WT over-expressing cell line, and p53-S149A mutant over-expressing hepatocytes. For each condition, 20–30 million cells were grown. To study the role of p53 gluconeogenic metabolic flux in vitro, cell medium from untreated cells were changed to KHH, in the presence of pyruvate-1-¹³C (110 mg/L of media) (Merck, 490709). Cells were incubated for 24 h, washed in PBS twice, and harvested in the same buffer. Cell metabolic extraction was performed as explained⁶⁹. The hydrophilic metabolites were resuspended in 300 µl of D₂O with 0.11 mM of Sodium trimethylsilylpropanesulfonate (DSS, internal reference). All NMR experiments were recorded at 298 K on a Bruker 600 MHz (12 T) Avance III spectrometer equipped with a BBO probehead. For each sample, three different experiments were collected: (I) 1D ¹H p3919gp with water signals suppression using a binomial 3-9-19 pulse with echo gradient pair, (II) 1D ¹³C zgpg30 with power-gated decoupling, and (III) phase-sensitive gradient-enhanced 2D 1H-13C HSQC (hsqcetgp). Chemical shift assignment was performed using standard compounds. Peak integration and quantification were done using TopSpin 4.0.7 (Bruker Biospin GmbH) and in-house MatLab scripts.