To begin the cell culture, prepare the DMEM culture medium and solutions required for cell culture. Seed 1 x 10 to the 6th HEK293 cells in a 60-millimeter plate or a 6-well plate per treatment condition plus control. The next day, treat the cells with DNA-protein crosslinks, or DPCs, inducers of choice.
To induce DPCs, their ubiquitylation and SUMOylation, collect the cells at 20, 60, and 180 minutes, or two hours in case of non-enzymatic DPCs. To induce the PARylation of TOP1-DPCs, pre-treat the cells with 10 micromolar poly-ADP-ribose glycohydrolase, or PARG inhibitor PDD00017273, for one hour before co-treating with 20 micromolar camptothecin for 20, 60, and 180 minutes.