D glucose 13c6

The cells were inoculated into 5 ml of SD medium and were cultured at 25°C overnight. To enter the log-phase, an aliquot of precultured cells was resuspended in 10 ml of SD medium and were cultured at 25°C for 3 h. To incorporate ¹³C into cellular metabolites, 0.4% D-glucose-¹³C₆ (Sigma-Aldrich, St. Louis, MO, USA) and 1.6% of natural D-glucose were used as carbon sources in the SD medium. The cells were harvested and suspended in 10 ml of SD medium containing ¹³C-glucose and were subsequently cultured at 25°C until an OD₆₀₀ of 0.5 was reached.

P. aeruginosa PA14 was grown in M9 minimal media (2 mM MgSO₄, 0.1 mM CaCl₂, 0.4 % weight/volume (^w/_v) glucose, 47.9 mM Na₂HPO₄, 22 mM KH₂PO₄, 8.2 mM NaCl, 18.7 mM NH₄Cl), with the only carbon source being d-glucose-¹³C₆, (Sigma-Aldrich, Munich, Germany). To ensure all carbons were isotopically labeled (¹³C), cells were maintained in M9 minimal media containing ¹³C labeled glucose for at least two passages. P. aeruginosa was grown to OD₆₀₀ ≥ 1.5 at 37 °C and cells were harvested by centrifugation and washed 1× with 0.9 % saline.

Cells from each experimental group were plated in three wells of 6-well plates and analyzed independently as follows. The culture medium from cells undergoing reprogramming at 10 dpi was replaced with DMEM without glucose (Nacalai Tesque) supplemented with 4.5 g/L D-glucose-¹³C₆ (Sigma-Aldrich), followed by incubation with 5% CO₂ for 1 h at 37 °C. Then, the medium was aspirated, and the cells were washed in ice-cold PBS three times with chilling, followed by snap freezing in liquid nitrogen. The plates were stored at − 80 °C until metabolite extraction. The methods used for metabolite extraction and cytosolic fractionation to remove mitochondria from cell lysates have been described previously [22 , 23 (link)]. See Additional file 5 for further details.

MEFs were exposed to torin1 (475991, Merk Millipore, Burlington, MA, USA) and staurosporine (81590, Cayman Chemical, Ann Arbor, MI, USA), which had been pre-dissolved in dimethylsulfoxide (DMSO). MEF cells were seeded in Permanox 1-well Lab-Tek chamber slides from Nunc, Thermo Scientific (Rochester, New York, NY, USA) in Dulbecco’s Modified Eagle Medium (DMEM) (Life Technologies, Carlsbad, CA, USA) supplemented with 10% Fetal Bovine Serum (FBS) and 1% Penicillin-Streptomycin (Life Technologies, Carlsbad, CA, USA). Internal standards (IS) for hippuric acid-(phenyl-¹³C₆), L-lysine-¹³C₆−¹⁵N₂, leucine-5,5,5-D₃, D-glucose-¹³C₆, glyceryl tri-(palmitate-1-¹³C), and cholic acid-2,2,4,4-D₄ were purchased from Sigma-Aldrich (St. Louis, MI, USA). Caffeine-¹³C₃ was acquired from Cerilliant Corporation (Round Rock, TX, USA), 18:1-D₇ lyso-phosphatidylethanolamine (LPE), 18:1-D₇ lyso-phosphatidylcholine (LPC) from Avanti Polar Lipids (Birmingham, UK), octanoyl-L-carnitine-(N-methyl-D₃), ceramide [d18:1/18:1-(9Z)-¹³C₁₈] and L-phenylalanine-¹³C₉−¹⁵N from Cambridge Isotope Laboratories (Andover, MA, USA). Additional materials and chemicals are described in Supplementary Materials Section S1.