Metabolic Analysis of K562 Cells

K562 cells were cultured and polar cell extracts were prepared as described before [4 (link)]. For tracer-based metabolic analysis, standard glucose- or glutamine-free RPMI-1640 media (Gibco) was used and substituted with 2 g/l [1,2-¹³C]glucose or 300 mg/l [3-¹³C]glutamine (Isotec, Sigma), respectively. 5 × 10⁷ exponentially growing K562 cells per control or treatment were pelleted by centrifugation at 8000g for 5 min and resuspended in the relevant media with BaP or solvent controls and incubated for 24 or 3 h at 37 °C and 5 % CO₂ in a humidified incubator. For BaP treatment, bezafibrate 0.5 mM and medroxyprogesterone acetate 5 μM (Sigma) or the equivalent concentrations of DMSO and ethanol solvent control were added to the media at T = 0 h.
For 3-h labelling of 24-h drug treatments, media was exchanged for the last 3 h with media supplemented with labelled glucose or glutamine plus BaP or solvent control. All samples were dissolved in 100 mM phosphate buffer with 10 % D₂O and 500 μM Trimethylsilylpropanoic acid (TMSP) added.