Fig. 2

Impaired CPD excision upon ASH1L depletion. a Visualization of H3K4me3 in ASH1L-depleted HeLa cells. Whole cell lysates were analyzed by immunoblotting using antibodies against the indicated forms of H3. UV radiation was at 10 J m^–2. b Quantification of H3K4me3 levels normalized to total H3 (n = 6 independent experiments). Error bars show s.e.m. (one-sample t-test). c Initial CPD formation after UV irradiation. HeLa cells were transfected with siASH1L or siNC, UV-irradiated (10 J m^–2) 48 h later and immediately collected for CPD analysis. Immunoassay absorbance, providing a measure of CPDs, were not affected by the ASH1L depletion (n = 3, each experiment with 4 replicates; two-tailed t-test). d Excision of CPDs in HeLa cells treated 48 h before UV radiation (10 J m^–2) with siRNA targeting ASH1L or SETD2, compared to transfections with siNC (n = 5, each experiment with 4 replicates, two-tailed t-test). e Excision of CPDs in HeLa cells transfected 48 h before UV radiation (10 J m^–2) with siASH1L, siASH1L_2 or siSETD2, compared to siNC. CPDs were measured immediately after UV treatment and after a 24-h repair period (n = 3 with 4 replicates each, two-tailed t-test). f Excision of 6–4PPs in HeLa cells transfected 48 before UV radiation (10 J m^–2) with siRNA targeting ASH1L in comparison to siXPA and siNC (n = 3 with 4 replicates each, two-tailed t-test). g Immunofluorescence detection of CPDs in cells transfected with siASH1L or siNC, 48 h before UV radiation (100 J m^–2) through 5-µm filter pores. DNA was stained with DAPI. The quantification of nuclei positive for CPD spots is based on a minimum of 100 cells (n = 3 independent experiments, two-tailed t-test). Scale bar = 15 µm. Representative wide-field views of cells are shown in Supplementary Fig. 6