Cell Cycle Progression and DNA Damage Assay

Cell cycle progression of replicating cells was assayed using a modified method, as previously described [30 (link)], with the following modifications; cells were seeded and the following day they were synchronized with 24 h treatment of 6 µM aphidicolin, released into media containing 10 µM 5-Ethynyl-2’-deoxyuridine (EdU) (Sigma-Aldrich, #1T511285) for 45 min, followed by treatment with 10 µM cisplatin alone or in combination with 5 µM PFKB3i for 6 h or 16 h. Post blocking, cells were incubated with primary antibody γH2AX-Ser139 (Millipore, #05-636, 1:500) overnight at 4 °C. Cells were washed twice with 1% BSA/PBS solution and incubated with secondary antibody goat anti-mouse Alexa 405 (1:50, #A31553, Invitrogen, Carlsbad, CA, USA) for 30 min at 37 °C followed by Click-iT reaction.