Figure 4. XPC release is SUMO and K63-ubiquitylation dependent.

(a) Top panel: representative pictures of co-localization of XPC with CPD at LUD in U2OS cells transfected with the indicated siRNA's 30 min or 4 h after local UV irradiation (60 J m⁻²) are shown. Scale bar, 5 μm. Bottom panel: quantification of XPC co-localization with the damage marker CPD. (n≈50 cells containing a LUD were scored per sample in three independent experiments; error bars are the mean±s.d.). The immobilized fraction of XPC–GFP (b) or ERCC1–GFP (c) as determined by FRAP analysis in mock or UV-treated (10 J m⁻²) cells depleted by siRNA of UBC9 or UBC13 (n=40 from two experiments; error bars are the mean±2 × s.e.m.). (d) HeLa/FLAG-SUMO2 cells were transfected with plasmids expressing WT or K8R XPC–GFP, then left untreated or incubated with doxycycline (DOX) to induce FLAG-SUMO2 expression. One hour after UV exposure (16 J m⁻²), cells were lysed under denaturing conditions, and XPC SUMOylation was analysed by immunoblotting of FLAG IPs with GFP antibody. (e) The immobilized fraction of WT XPC–GFP or K8R XPC–GFP as determined by FRAP analysis in mock or UV-treated (10 J m⁻²) cells (n>40 from three experiments; error bars are the mean±2 × s.e.m.). (f) Cells stably expressing WT XPC–GFP or K8R XPC–GFP were locally irradiated using a 266 nm UV-C laser. GFP fluorescence intensity at UV-C laser-induced LUD was measured over time using live-cell confocal imaging and quantified to predamage intensity set at 1 at t=0 (n>25 cells per sample, measured in two independent experiments; error bars are the mean±s.e.m.). (g) XPC deficient XP4PA cells stably expressing WT XPC–GFP of K8R XPC–GFP were locally UV-irradiated (60 J m⁻²) and immunostained for endogenous XPB, ERCC1 and XPF proteins 30 min later. The percentage of co-localization with GFP–XPC at LUD is plotted in the graph (n>100 cells containing a LUD were scored in at two independent experiments; error bars are the mean±s.d.).