Fig. 3: RAD18 3A mutation impairs PCNA monoubiquitination and cell survival after UV irradiation.

A HEK293T cells transfected with SFB-RAD18 were irradiated with UV (15 J m⁻²) and harvested at different time points later. The cell lysates were denatured and immunoprecipitated with anti-S beads followed by immunoblotting with O-GlcNAc and Flag antibodies. Chromatin fractions of HEK293T cells transfected with SFB-RAD18 (B) or incubated with glucose or not (C) were extracted after UV (15 J m⁻²) irradiation followed by immunoblotting with indicated antibodies. RAD18 O-GlcNAcylation promotes PCNA monoubiquitination. RAD18+/+ or RAD18-/- HEK293T (D) and U2OS (E) or OGT-depleted RAD18-/- HEK293T (F) cells were transfected with WT or 3A RAD18, and irradiated with UV (15 J m⁻²). The chromatin fractions were harvested and immunoblotted using indicated antibodies. G Clonogenic survival assays in RAD18-knockdown U2OS cells transfected with empty vector, WT or 3A Flag-RAD18 after UV irradiation. Cells were irradiated with indicated doses of UV and further incubated in medium supplemented with 0.4 mM caffeine for 7–10 days. Surviving fraction was expressed as a percentage of mock-treated cells. The representative curve is shown. Error bar: s.d., n = 3. The protein levels of RAD18 were detected by immunoblotting.