ZRF1 and RING1B contribute to GG-NER. (A) RING1B and ZRF1 knockdown fibroblasts are defective in UDS after UV irradiation. UDS was measured by EdU incorporation after UV treatment in MRC5 fibroblasts with shRNA-mediated knockdown of the indicated proteins. XPA fibroblasts were used as a positive control. Values are given as mean ± SEM. Data were acquired from three independent experiments (150–300 nuclei per sample). (B) RING1B and ZRF1 knockdown fibroblasts are defective in the removal of CPDs. The CPD removal was analyzed in MRC5 fibroblasts after knockdown of the indicated proteins in MRC5 fibroblasts and in XPA fibroblasts. Cells were irradiated with 10 J/m² and fixed immediately or 24 or 48 h after irradiation and stained with CPD antibodies. The relative fluorescence intensity was determined. Values are given as mean ± SEM. Data were acquired from three independent experiments (100–200 nuclei per sample). (C) MRC5 fibroblasts were treated with lentiviral particles containing the respective shRNA. Knockdown of the proteins levels was analyzed 48h after infection by Western blotting and incubation with the indicated antibodies. (D) C. elegans knockout mutants for ZRF1 (dnj-11) and RING1B (spat-3) show increased sensitivity toward UV irradiation. Late-L4 larval wild-type worms and the indicated mutants were irradiated with UV light at different doses, and the relative viability was determined by comparing hatched versus dead embryos (unhatched eggs). Values are given as mean ± SEM (n = 3). (E) C. elegans knockout mutants for dnj-11 and for spat-3 show only weak developmental arrest upon somatic UV irradiation. L1 larval worms were irradiated with UV light at different doses. Relative larval-stage stalling was determined after 60 h by using a large particle flow cytometer (BioSorter platform; Union Biometrica), assaying at least 1,000 worms per condition.