Fig. 1.

ZNF506 localizes to DNA double-strand break in an MDC1-dependent manner. a, b Downregulation of ZNF506 using shRNAs leads to persistent DNA double-strand breaks. a Representative images of γ-H2AX foci (green) in U2OS cells after the indicated treatments and indicated time following 2 Gy irradiation (IR). Nucleus is stained with DAPI (blue). b Quantification of γ-H2AX foci in U2OS cells after the indicated treatment and time following IR. c ZNF506 localizes to the DNA double-strand break site in U2OS I-SceI cells where one DNA double-strand break is induced per cell using triamcinolone acetonide (red). This ZNF506 focus (green) overlaps with γH2AX (blue). The yellow box locates the site of the cut. The white circle outlines the nucleus. d ZNF506 forms radiation-induced puncta that overlaps with γ-H2AX in U2OS cells. Cells were stained for γ-H2AX foci (green). Nucleus is stained with DAPI (blue). Endogenous ZNF506 shows diffuse nuclear staining without DNA damage but forms discrete nuclear foci that overlaps with γ-H2AX upon treatment with 2 Gy IR (red). e, f MDC1 is required for recruitment of ZNF506 to double-strand break sites. U2OS cells in which MDC1 was knocked out using CRISPR were exposed to 2 Gy IR and stained for the indicated foci. Nucleus is stained with DAPI (blue). e Quantification of ZNF506 foci in U2OS cells wild-type or knockout for MDC1 and treated as indicated. f Representative images of the indicated foci in U2OS cells an hour after 2 Gy IR. g Endogenous ZNF506 interacts with MDC1 upon DNA damage. Shown are the representative data (mean ± SEM) from three independent experiments in b and e. **p < 0.01 by ANOVA comparing the knockdown groups to control at each time point in b, and control to knockout with each treatment condition in e. Representative images of three independent experiments are shown in a, c, d, and f. Scale bars, 10 µm. Representative western blots in g are provided from three biologically independent experiments. Unprocessed blots are provided in Supplementary Fig. 5