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. 2020 Nov 18;19(24):3437–3457. doi: 10.1080/15384101.2020.1843777

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This work was authored as part of the Contributor’s official duties as an Employee of the United States Government and is therefore a work of the United States Government. In accordance with 17 U.S.C. 105, no copyright protection is available for such works under U.S. Law.

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Figure 1. — A hydrophobic-motif-dependent aggregative activity of Cep63 and Cep152. (a), 3D-SIM analysis for immunostained HEK293 cells co-infected with adenoviruses expressing mCherry-Cep63 and mGFP-Cep152. The location of endogenous centrioles is marked by anti-Cep192 signals. DAPI, chromosomal DNA stained by 4',6-diamidino-2-phenylindole. Arrowheads, a pair of endogenous centrioles. Asterisk, a ring-like aggregate found away from the two centrioles indicated by arrowheads. Note the cooperative generation of pericentrosomal aggregates by mCherry-Cep63 and mGFP-Cep152. Boxes, areas of enlargement. Scale bar, 2 µm. (b), CLEM analysis for HEK293 cells expressing mCherry-Cep63 and mGFP-Cep152. The endogenous centriole is marked by anti-Cep192 signal. Electron micrographs show an amorphous matrix-like aggregate around a centriole (asterisk). Box, area of enlargement. (c–e), Confocal microscopy, quantification, and immunoblotting analyses of HEK293 cells infected with adenoviruses expressing either WT Cep63 and Cep152 or their respective 4A and 5A hydrophobic mutants. Boxes in (c), areas of enlargement. Quantification in (d) was carried out by determining the total signal intensities (relative) of 12 randomly chosen fields (224.92 μm x 224.92 μm/field) from each sample using 2.5D intensity plots generated by the ZEN black software. Each microscopic field contains >50 cells. The data in (d) are shown in mean ± s. d. Exemplary intensity profiles for 2.5D plots are shown in Fig. S1B. ****, P < 0.0001 (unpaired two-tailed t-test). Immunoblots in (e) show the levels of WT and mutant forms of Cep63 and Cep152 expressed in the transfected cells. CBB, Coomassie Brilliant Blue–stained membrane. (f–h), 3D-SIM, quantification, and immunoblotting analyses of cells expressing endogenous promoter (Pendo)–controlled mCherry-Cep63-sil and mGFP-Cep152-sil and depleted of endogenous Cep63 and Cep152 by RNAi. Arrowheads in (f) indicate active, dot-state Plk4 signals. The data in (g) are shown in mean ± s. d. *, P < 0.05, ****, P < 0.0001 (unpaired two-tailed t-test). CBB, Coomassie Brilliant Blue–stained membrane