Figure 1.

PLK4 localizes at discrete sites on the wall of the parent centriole. (A) Widefield immunofluorescence of PLK4 localization in DMSO or centrinone-treated RPE1, DLD1, U2OS, and HeLa cells using PLK4 #1 antibody. The total n for each experiment was ≥10 over three biological replicates. (B) Schematic showing the localization of PLK4 (green) on the CEP152 (gray) ring. Quantification of the PLK4 localization pattern in DMSO or centrinone-treated RPE1, DLD1, U2OS, and HeLa cells using the PLK4 #1 antibody. (C) Representative U-ExM images of PLK4 (magenta), CEP152 (cyan), and acetylated α-tubulin (green) in unduplicated and duplicated parent centrioles. The number of PLK4 sites observed in each image is indicated. (D) Schematic representation of the analysis pipeline used to quantify the amount of PLK4 localized at individual sites on parent centrioles. An intensity line scan of PLK4 was performed around the wall of the parent centriole and the AUC was calculated for each site. Sites were ordered based on decreasing AUC values and plotted to represent the percent of PLK4 protein occupying each site. Peaks that contained >2% of centriolar PLK4 and had an intensity above 1,500 were considered as a PLK4 site. (E) Graph showing the amount of PLK4 localized at individual sites on parent centrioles. All representative images are scaled independently to best represent phenotypes. The asterisk denotes the procentriole. Data are represented as mean ± SEM. Scale bar = 1 µm for widefield immunofluorescence images and 250 nm for U-ExM images.