Fig. 4

Plk4 containing the phospho-PC3 motif is stable and colocalizes with STIL and Sas6 along the length of an elongated procentriole. a Immunoblotting analyses of U2OS cells stably expressing the indicated constructs treated with siRNA (siGL or siβTrCP) or cycloheximide (CHX). Total lysates were treated with λ phosphatase (PPase) to convert all phosphorylated, slow-migrating Plk4 forms into a fast-migrating form (arrows). Numbers, relative signal intensities. Quantification of relative signal intensities is shown in mean ± s.d. (n = 3 independent experiments). b 3D-SIM analysis and quantification of immunostained HEK293T cells stably expressing the indicated EGFP-Plk4 constructs and transfected with Flag-βTrCP. Bar, 1 μm. Enlarged images (bar, 0.2 μm) for dotted boxes are shown. The fraction of cells with or without colocalized Flag-βTrCP signals was quantified (right) (mean ± s.d., n = 3 independent experiments). ****P < 0.0001 (unpaired two-tailed t test). Note that elongated (inside dotted boxes) and dot-state (arrowheads) Plk4 signals are colocalized with STIL, but not with βTrCP, while the ring-state Plk4 (yellow arrow) is colocalized with Cep152 and βTrCP. c 3D-SIM analysis of U2OS cells immunostained with anti-Cep152, anti-Plk4, and Alexa Fluor 594-conjugated anti-Plk4 pSSTT antibodies. Images representing different stages from G1 to S/G2 are shown. Bar, 0.5 μm. See the confocal images in Supplementary Fig. 4e that demonstrate the specificity of the Alexa Fluor 594-conjugated Plk4 pSSTT antibody. Note that the Plk4 pSSTT epitope preferentially associates with the dot state (arrows) and elongated (barbed arrow) Plk4 signals rarely observed at native centrosomes. d Quantification of the relative intensities of Plk4 pSSTT, STIL, STIL pS1108, and Sas6 signals was carried out using confocal images from immunostained U2OS cells. Relative fluorescence intensities (mean ± s.d.) are shown. n = 90 (WT), 90 (KM), and 90 (CP) for Plk4 pSSTT/Plk4, n = 90 (WT), 91 (KM), and 97 (CP) for STIL/Plk4, n = 132 (WT), 103 (KM), and 142 (CP) for STIL pS1108/Plk4, and n = 90 (WT), 91 (KM), and 97 (CP) for Sas6/Plk4 with each sample pooled from three independent experiments. Numbers denote mean relative intensities. Representative images are shown in Supplementary Fig. 4f–h. ****P < 0.0001 (unpaired two-tailed t test). Numbers denote mean values. e 3D-SIM analysis of immunostained U2OS cells stably expressing EGFP-Plk4 CP. Bars, 5 μm. Enlarged images (green bars, 1 μm) for dotted boxes are shown. Quantification of images is shown in mean ± s.d. (n = 3 independent experiments). Source data are provided as a Source Data file for a, b, d, e