Figure 6. PLK1-dependent phosphorylation of RAD9 is required for proliferation under replicative stress.

(A) Cell growth was monitored using the Presto-Blue Cell viability reagent (see Materials and methods). U2OS cells stably expressing RAD9-WT-mH (WT), RAD9-S291A/T292A-mH (T292A), and RAD9-T313A-mH (T313A) from the FRT locus, and the host U2OS T-REx cells (Mock) were used. (B) The cellular viability in the presence of hydroxyurea was monitored by a colony formation assay (see Materials and methods). U2OS cells stably expressing RAD9-WT-mH (RAD9-WT) and RAD9-T313A-mH (RAD9-T313A) from the FRT locus, and the host U2OS T-REx cells (Mock) were used. See also Figure 6—figure supplement 1. Reduced cell proliferation by expression of non-phosphorylatable RAD9 in telomerase-positive HEK293A cells.

Figure 6—figure supplement 1. Reduced cell proliferation by expression of non-phosphorylatable RAD9 in telomerase-positive HEK293A cells.

HEK293A T-REx cells expressing RAD9-WT, RAD9-T292A or RAD9-T313A were grown in 96 well plates in the presence of 0.2 mM or 0.4 mM hydroxyurea for 4 days. siRNA (s11719: Ambion) was transfected 48 hr before starting the incubation with HU, to knock down the endogenous RAD9. Viable cells were measured using Presto-Blue (Thermo Fisher), and relative values against that obtained from cells incubated without hydroxyurea are indicated. Averages and standard deviations obtained from the values from the three slots are shown as a graph.