Figure 6
DYRK1A co-localizes with RNF169 at DSBs. (A) HeLa cells were transiently transfected with plasmids for the indicated proteins (GFP-DYRK1A and FLAG-RNF169 versions) and 36 h later, they were subjected to IR (3 Gy; +IR) or left untreated (-IR). Cells were fixed after a 1 h recovery and immunostained with the Abs indicated, counter-staining the nuclei with DAPI (blue). Scale bar, 10 µm. Some of the images include an inset to show the overlap in staining. (B) HeLa cells expressing FLAG-RNF169 or FLAG-RNF169∆1 (see Fig. 4A) were transfected with a plasmid encoding GFP-DYRK1A. Following 10 Gy irradiation, the cells were pre-extracted using 0.5% Triton X-100 prior to PFA fixation. The cells were subsequently immunolabeled for RNF169 (anti-FLAG) or γH2AX 1 h after recovery, and those cells expressing DYRK1A were detected by direct GFP fluorescence. Only irradiated cells are shown (+IR). (C) The U2-OS mCherry-Fok1 reporter cell line was used to detect recruitment of endogenous DYRK1A to DSBs. The DSB site was detected by direct mCherry fluorescence, while the DYRK1A Ab-C1 was used to detect endogenous DYRK1A. Nuclei were counterstained with DAPI. (D) DYRK1A was allowed to interact with RNF169 and DCAF7 from untreated or irradiated (3 Gy) HeLa cells and collected in RIPA-buffer 1 h after recovery. (E) HeLa cells ectopically expressing FLAG-DYRK1A (WT) or a kinase-mutant version (KR) were irradiated (3 Gy), and subjected to immunofluorescence staining 1 h later with Abs to FLAG (green) and 53BP1 (red). The nucleus of the cells was counterstained with DAPI (blue). Scale bar, 10 µm. (F,G) HeLa cells expressing the different FLAG-tagged RNF169 proteins were irradiated (3 Gy) and processed for immunofluorescence analysis with anti-FLAG and 53BP1 Abs 1 h after recovery. Cells with more than 10 foci were quantified as positive. The graph shows the percentage of cells positive for RNF169 (F) or 53BP1 (G) in control cells and after IR (mean ± SEM of two independent experiments). See Fig. S7B for the analysis of the interaction between DYRK1A and the RNF169 proteins.
