A5502

Metabolite extraction and quenching were performed on plate with 1.5 mL ice cold extraction medium (90% methanol, 10% water) containing 1 µg/mL ribitol (A5502, Sigma Aldrich), phenyl β-d-glucopyranoside (292710, Sigma Aldrich), isoguanosine (sc-207768, Santa Cruz, Dallas, TX, USA), d4-succinate (293075, Sigma Aldrich) and methyl-tyrosine (M8131, Sigma Aldrich) as the internal standard. Cells were detached on ice by using a cell scraper, transferred into screw-cap tubes prefilled with 300 mg glass beads (G4649, Sigma Aldrich) and immediately frozen in liquid nitrogen until homogenization. Cells were homogenized using a Precellys tissue homogenizer (P000669-PR240-A, Bertin instruments, Montigny-le-Bretonneux, France) at −10 °C. Three cycles of homogenization for 15 s at 6500 rpm were applied with 10-s breaks in between cycles. Samples were then centrifuged (20,000× g) for 10 min at 4 °C to remove cell debris and protein precipitates, and 500 µL of each supernatant was transferred into two new reaction tubes for GC-MS and LC-MS analyses. Finally, extracts were dried using a vacuum rotator (Eppendorf, Hamburg, Germany) and flushed with nitrogen. The DNA content in the extracts was measured using NanoDrop 1000 (Thermo Fisher Scientific).

Cells of 3x10⁵/well 6 well plate or 2x10⁴/well 96 well plate were seeded in triplicate in the growth medium unless specified otherwise. The following day, the medium was supplemented with ribitol (A5502 Sigma, St. Louis) or D-ribose (R7500 Sigma) or xylitol (X3375 Sigma) at a concentration of 10 mM, unless otherwise noted, and the cells were grown in the supplement for 3 days. Plates were washed with PBS and cells were processed for analysis by FACS, metabolomics, immunocytochemistry, and western blot.