Centriolar satellites are sites of translation of centrosomal and ciliary proteins in vertebrate cells. (A) Schematic of the Puro-PLA. Puro-PLA combines puromycin labeling with proximity-dependent ligation to make labeling specific for a particular protein of interest, here PCNT. (B) Puro-PLA reveals nascent PCNT in proximity to centriolar satellites visualized using PCM1-GFP. To exclude random colocalization/proximity, distribution was compared with randomized controls (see Materials and methods). N = 109 cells. (C) Immunofluorescence micrographs and quantitation of centriolar satellite protein signal in control and puromycin-treated HeLa cells. Centrosomal and ciliary proteins PCNT, CEP290, CDK5RAP2, and CEP131 are significantly depleted of their cytoplasmic localization upon puromycin treatment, whereas their centrosome localization persists (centrosomes identified using anti-γ-tubulin countermarker, not shown), suggesting the former represents newly synthesized protein. In contrast, foci of PCM1, OFD1, and MIB1 remain, although now dispersed throughout the cytoplasm. >100 cells were analyzed per condition. Mean ± SD are displayed. A Mann–Whitney test was used to assess statistical significance; ****P < 0.0001. (D) smFISH combined with immunofluorescence microscopy in control and puromycin-treated HeLa cells. PCNT mRNA localizes in the vicinity of PCM1 independently of ongoing translation. To exclude random colocalization/proximity, distribution was compared with randomized controls (see Materials and methods). Error bars are the mean ± SD. N = 77 cells (control), 102 cells (puromycin). Scale bars, 5 µm (B and D), 10 µm (C), 1 µm (insets). See also Fig. S5.