Fig. 3.

AKT inhibition impairs PCNA ubiquitylation only in the context of replication stress. a U2OS cells were pre-treated overnight with the indicated kinase inhibitors: PI3K inhibitor (LY294002 50 µM), ATR inhibitor (VE-821 1 µM), ATM inhibitor (KU-55933 1 µM), DNA PKcs inhibitor (NU7026 20 µM), and AKT inhibitor (C11 1 µM). Cells were then submitted to UV irradiation (15 J/m²), and 12 h in the presence of the inhibitors at the same concentrations, samples were processed by WB for the quantification of ubi/PCNA. b U2OS cells were treated in parallel and in combination of suboptimal doses of the AKT inhibitor C11 (0.1 µM) and the optimal dose of the DNA PKcs inhibitor NU7026 (20 µM). Twelve hours after UV irradiation (15 J/m²), samples were processed by WB for the quantification of ubi/PCNA. c U2OS cells were treated overnight with the optimal dose of each kinase inhibitor (LY294002 50 µM, NU7026 20 µM, and C11 1 µM) followed by sample processing for WB in unperturbed conditions. d U2OS cells were transfected with 75 nM of siRNA against USP1. Forty-eight hours later, cells were treated with C11 1 µM and after 12 h, samples were processed for quantification of PCNA ubiquitylation by western blot