DDB2-dependent recruitment of ALC1 to UV lesions. (A) NHF cells were locally UV irradiated (100 J/m²), fixed after the indicated time, and stained with an antibody recognizing ALC1 or TFIIH. ALC1 colocalizes with the damage marker TFIIH. (B) XP-A cells stably expressing GFP-ALC1 were infected with the indicated shRNA. The cells were UV damaged using UV-C (266 nm) laser irradiation. GFP fluorescence intensities at the site of UV damage were measured by real-time imaging until they reached a maximum. Assembly kinetic curves were derived from at least six cells for each protein. Error bars indicate SEM. (C) Clonal survival of UV-irradiated NHF cells expressing shControl or shALC1 and XPA cells. The percentage of surviving cells is plotted against the applied UV-C dose (J/m²). The results are from three independent experiments. Error bars indicate SD. Bars, 20 µm. (D) NHF cells expressing shControl or shALC1 RNAi or treated with 10 µM PARPi were irradiated with 10 J/m² UV-C, fixed immediately, at 8 or 24 h after UV treatment, and stained with anti-CPD antibody (*, P < 0.05, analysis of variance). (E) NHF cells expressing shControl or shALC1 or treated with 10 µM PARPi were irradiated with 10 J/m² UV-C, fixed immediately, at 1 or 2 h after UV treatment, and stained with an anti–6-4PP antibody. The total fluorescence intensity of the nucleus was quantified and divided by the surface area, resulting in a specific fluorescence intensity expressed in arbitrary units. Values are the result of three independent experiments (100 cells per time point). (D and E) Error bars indicate SD.