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. 2017 Apr 27;8:15164. doi: 10.1038/ncomms15164

Figure 1. Cyclin A mediates the localization of Cdc6 on the proximal side of the centrioles.

Figure 1

(a) Cdc6 localizes on the proximal side of centrioles during S and G2 phases. Immunofluorescence showing localization of endogenous Cdc6 (green) and centrin1 (red) in U2OS cells. We defined cells with four contiguous centrin-positive dots as S/G2 phase cells, and cells with two pairs of very distant centrin-positive dots as late G2 phase cells. (b) Cdc6 or WB mutant, but not WA mutant, localizes on centrioles. U2OS cells were transfected with GFP-tagged Cdc6 WT, WA or WB mutant and stained with a centrin1 antibody. (c) Cdc6 directly binds cyclin A in vitro. Sepharose beads coupled with purified GST or GST-tagged Cdc6 were incubated with in vitro transcribed and translated HA-tagged cyclin A and analysed by western blotting using a HA antibody. The loading of GST and GST-tagged Cdc6 proteins are shown by Coomassie blue staining. (d) Cytoplasm and centrosomal localization of Cdc6 requires the interaction between Cdc6 and cyclin A. U2OS cells were transfected with GFP-tagged Cdc6 Δcy and stained with a γ-tubulin antibody. (e) Interactions between Cdc6 and centrosomal localization signal (CLS) domain, or CLS deletion mutant (ΔCLS) of cyclin A are reduced compared to WT cyclin A. HEK293 cells were co-transfected with Myc-tagged Cdc6 and GFP-tagged cyclin A WT, ΔCLS or CLS truncate. The total cell extract was used for immunoprecipitation with a GFP antibody and western blotting with Myc and GFP antibodies. (f) The centrosomal localization of cyclin A is required for the centrosomal recruitment of Cdc6. U2OS cells expressing GFP-tagged truncates of cyclin A (amino acids 1-200, CLS or CLS mutant DWVE-A) were stained with Cdc6 and γ-tubulin antibodies. (g) Quantitation of cells with centrosomal Cdc6 in f. Approximately 300 cells were counted per sample, and three independent experiments were conducted. Data are presented as means±s.d. **P<0.01; N.S., no significant difference (Student's t-test). DNA was stained with DAPI. Scale bars, 10 μm. Insets in a,b,d and f are high-magnification views of the regions indicated in the low-magnification images.