Fig. 2: CHAF1A is required for the TLS pathway.

A DNA fiber assay was used to measure DNA replication in inducible CHAF1A knockdown KYSE510 cells. Schematic of alternative IdU/CldU pulse-labeling protocol to evaluate fork status. Representative images of DNA fiber assays are shown. B Image J was used to calculate the percentage of elongation forks in each field of view (n = 10). Data represent means ± SEM from three independent experiments. ^**P < 0.01; t-test. C IdU and CldU staining in a single DNA fiber were presented as representative images. Data are plotted in D (n = 100). Data are representative of at least three independent experiments. ^**P < 0.01; t-test. E CHAF1A-knockdown KYSE510 cells were treated with or without 100 J/m² UV for 1 h. Cell fractionation was performed to detect indicated antibodies. F CHAF1A-knockdown KYSE510 cells were treated with or without 100 J/m² UV for 1 h. PLA assays was used to detect the interaction between PCNA and TLS polymerases (Pol ι, REV1, Pol η, and Pol κ) (scale bar, 5 μm), and (G) each spot represents the number of PLA foci in an individual nucleus (n = 100). Data represent means ± SEM from three independent experiments. ^**P < 0.01; one-way ANOVA. H and I Colony formation assay for KYSE510 cells or A549 cells with treatment of the indicated dose of UV (n = 3). Data represent the mean ± SEM from three independent experiments. ^*P < 0.05; one-way ANOVA.