(a-g) HCT116 cells were sequentially incubated with CldU and IdU in the presence of increasing concentrations of P22077, P5091, HBX19818 or DMSO as a control. Representative images of stretched DNA fibers visualized by IF for CldU (red) and IdU (green) are shown in (a). Fork rate (b-d) and percentage of new origin firing (e-g) were measured and quantified after the different treatments. All the experiments were repeated three times; the pool of the three experiments (fork rate) or the average (origin firing) is shown. * p<0.05; ** p<0.01; *** p<0.001, t- test.
(h) 293T-REx cells with inducible expression of OneStrep-FLAG-HA-USP7 were treated with 1 µg/ml Doxycycline (+) or EtOH as a control (-) for 1 day. Whole cell extracts were analyzed by WB with antibodies against USP7 or MEK2 as a loading control.
(i) DNA fibers were extracted from control (black bars) and doxycycline treated (grey bars) 293T-REx-USP7 cells treated with increasing amounts of P22077. Replication fork rates were measured in three independent experiments and the average normalized to the DMSO treated sample. *p<0.05, two-way ANOVA.