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Figure S5.
Further evidence for centriolar satellites as sites of translation in vertebrate cells, related toFig. 5. (A) Immunofluorescence micrographs of the centriolar satellite protein signal in control and cycloheximide-treated HeLa cells. The satellite client PCNT is significantly depleted of its cytoplasmic localization upon puromycin treatment, whereas its centrosome localization persists. In contrast, PCM1 signal remains, although it is now dispersed throughout the cytoplasm. (B) Immunofluorescence micrographs and quantitation of PCM1 and OFD1 distribution by Pearson’s correlation coefficient analysis in control and puromycin-treated cells from Fig. 5 C. PCM1 and OFD1 colocalize independent of translation. N = 125 cells (control), 147 cells (puromycin treatment). Mean ± SD are indicated (Student’s t test; *P < 0.05). (C) SmFISH combined with immunofluorescence microscopy shows PCNT mRNA colocalizing with the nascent PCNT protein in the cytoplasm. (D) Specificity of PCNT mRNA FISH and protein immunofluorescence signal confirmed by the absence of signal in PCNT KO cells. (E) Single-molecule fluorescence hybridization (smFISH) combined with immunofluorescence microscopy in HeLa cells. CEP290 mRNA localizes in the vicinity of PCM1. To exclude random colocalization/proximity, distribution was compared with randomized controls. N = 78 cells. (F) Single-molecule inexpensive fluorescence hybridization (smiFISH) combined with immunofluorescence microscopy in HeLa cells. Unlike PCNT and CEP290, RANBP10 mRNA does not localize proximal to PCM1. Distribution compared with randomized controls. N = 87 cells. (G) Immunofluorescence micrographs and quantitation of centriolar satellite signal in control and PCM1 siRNA–treated cells. Depletion of PCM1 largely eliminates cytoplasmic foci of OFD1, CEP290, CEP131, CDK5RAP2, while centrosomal signal remains. MIB1 signal is largely unaffected, although foci are now dispersed throughout the cytoplasm. Mean ± SD are indicated. N > 100 cells each condition. Statistical test to compare control and PCM1 depletions is t test with Welch’s correction (MIB1), and nonparametric Mann–Whitney test (others); ****P < 0.0001. Scale bars, 10 µm (A, B, and D–G), 5 µm (C), 1 µm (insets).

Further evidence for centriolar satellites as sites of translation in vertebrate cells, related to Fig. 5,. (A) Immunofluorescence micrographs of the centriolar satellite protein signal in control and cycloheximide-treated HeLa cells. The satellite client PCNT is significantly depleted of its cytoplasmic localization upon puromycin treatment, whereas its centrosome localization persists. In contrast, PCM1 signal remains, although it is now dispersed throughout the cytoplasm. (B) Immunofluorescence micrographs and quantitation of PCM1 and OFD1 distribution by Pearson’s correlation coefficient analysis in control and puromycin-treated cells from Fig. 5 C. PCM1 and OFD1 colocalize independent of translation. N = 125 cells (control), 147 cells (puromycin treatment). Mean ± SD are indicated (Student’s t test; *P < 0.05). (C) SmFISH combined with immunofluorescence microscopy shows PCNT mRNA colocalizing with the nascent PCNT protein in the cytoplasm. (D) Specificity of PCNT mRNA FISH and protein immunofluorescence signal confirmed by the absence of signal in PCNT KO cells. (E) Single-molecule fluorescence hybridization (smFISH) combined with immunofluorescence microscopy in HeLa cells. CEP290 mRNA localizes in the vicinity of PCM1. To exclude random colocalization/proximity, distribution was compared with randomized controls. N = 78 cells. (F) Single-molecule inexpensive fluorescence hybridization (smiFISH) combined with immunofluorescence microscopy in HeLa cells. Unlike PCNT and CEP290, RANBP10 mRNA does not localize proximal to PCM1. Distribution compared with randomized controls. N = 87 cells. (G) Immunofluorescence micrographs and quantitation of centriolar satellite signal in control and PCM1 siRNA–treated cells. Depletion of PCM1 largely eliminates cytoplasmic foci of OFD1, CEP290, CEP131, CDK5RAP2, while centrosomal signal remains. MIB1 signal is largely unaffected, although foci are now dispersed throughout the cytoplasm. Mean ± SD are indicated. N > 100 cells each condition. Statistical test to compare control and PCM1 depletions is t test with Welch’s correction (MIB1), and nonparametric Mann–Whitney test (others); ****P < 0.0001. Scale bars, 10 µm (A, B, and D–G), 5 µm (C), 1 µm (insets).

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