UNK is a centriolar satellite protein and promotes PCM1 and CEP131 localization. (A) UNK localizes to CEP131-positive centriolar satellites. SIM images of RPE-1-Tet-PLK4 Cetn2-GFP p53 WT cells showing UNK protein localization with endogenous and overexpressed PLK4 in S phase. UNK, magenta; CEP131, cyan; centrioles (centrin), green. Arrows indicate sites of UNK and CEP131 colocalization. Scale bar, 5 μm. Left insets scale bar, 0.5 μm. Right insets scale bar, 1 μm. (B) PLK4 overexpression promotes UNK localization to centrosomes in a time-dependent manner. Left panels: Confocal images of RPE-1-Tet-PLK4 Cetn2-GFP p53 WT cells showing UNK and CEP131 localization 6 and 12 h after PLK4 overexpression in S phase. UNK, magenta; CEP131, cyan; centrioles (centrin), green. Scale bar, 5 μm. Insets scale bar, 1 μm. Right top and middle panels: 8-μm radial fluorescence intensity and corresponding ratio quantification of UNK and CEP131 using centrin as the centrosome centroid. Right bottom—mean normalized centrosomal UNK and CEP131 fluorescence intensity based on binned central 2 μm. Graph values are expressed as the mean of three biological replicates of 25–30 cells per replicate and SD. P values were determined using one-way ANOVA with the Dunnett post hoc test. (C) Centriolar satellites are dispersed from centrosomes in siUNK cells with overexpressed PLK4. Left panels: SIM images of RPE-1-Tet-PLK4 Cetn2-GFP p53 WT cells showing centriolar satellites and their localization to the centrosomes in UNK-depleted cells with overexpressed PLK4 in S phase. PCM1, magenta; CEP131, cyan; centrioles (centrin), green. Scale bar, 5 μm. Insets scale bar, 1 μm. Right top panels: 8-μm radial fluorescence intensity of PCM1 and CEP131 using centrin as the centrosome centroid. Right, bottom left panels: 8 μm ratio quantification of PCM1 and CEP131 using centrin as the centroid. Right, bottom right panels: Centrosome PCM1 and CEP131 mean normalized fluorescence intensity based on binned central 2 μm. Graph values are expressed as the means of four biological replicates of 25–30 cells per replicate and SD. P values were determined using an unpaired two-tailed t test.