Nucleosides mix

Desmethylxestospongin B (dmXeB) (0–10 µmol/L), Ru360 (0–10 µmol/L, Sigma-Aldrich, San Luis, MO, USA), dimethyl α-ketoglutarate (5 mmol/L, Sigma-Aldrich), methyl pyruvate (5 mmol/L, Sigma-Aldrich), nucleosides mix (adenosine, cytosine, guanosine, uridine, and thymidine, 100 µmol/L each, Sigma-Aldrich).

Cells were plated in triplicate in six-well plates overnight (seeding density: 4T1—200 cells, EO771—1700 cells, MDA-MB-231—2000 cells, MDA-MB-468—2000 cells, DLD1—500 cells, RKO—2000 cells, HCT116—2000 cells, SW480—1000 cells, KPC—1700 cells). Cells were washed with PBS and the relevant experimental media change was performed and followed by irradiation. Media were refreshed every 4 days, and after 12–14 days of culture, colonies were fixed and stained (0.05% (w/v) Crystal Violet (Millipore), 1% formaldehyde, 1% methanol in 1XPBS). Images were taken on a GelCount (Oxford Optronix), colonies were counted using ImgeJ and the surviving fraction was determined (SF = plating efficiency treated cells/plating efficiency Ctr; plating efficiency = #colonies/#cells plated). Alternatively, the area under the curve was calculated and used for statistical testing. For the rescue experiments the experimental media were supplemented with the following compounds as indicated in the figure legends: 2 mM pyruvate (GIBCO), 2.5 mM or 5 mM reduced glutathione (sigma), nucleosides mix (Sigma; 30 µM citidine, 30 µM guanosine, 30 µM uridine, 30 µM adenosine, 10 µM thymidine), 5 mM N-acetylcysteine (NAC, sigma), 2 uM ferrostatin-1 (Fer-1), 50 uM Trolox or 100 nM liproxstatin-1 (Lip-1).