Figure S2.
Validation of CHMP4B mRNA localization at the MB. (A) Midbodies were purified from HeLa cells stably expressing GFP-MKLP1. Purified midbodies were plated on poly-L-lysine coated coverslips and analyzed by fluorescence microscopy. Arrows point to GFP-MKLP1 positive midbodies. Scale bar: 10 μm. (B and C) GFP-MKLP1 expressing HeLa cells were either mock-treated or treated with CHMP4B (B) or CEP55 (C) siRNAs. Cells were then incubated for 72 h, followed by mRNA isolation and RT-qPCR analysis using CHMP4B, CEP55, and GAPDH (control) specific primers. Data shown are the CHMP4B or CEP55 mRNA levels normalized against GAPDH mRNA derived from one qPCR analysis. (D and E) GFP-MKLP1 expressing HeLa cells were either mock treated (top two panels) or treated with puromycin for 1 h (bottom two panels) and subjected to staining with smFISH probes against CHMP4B mRNA. CHMP4B mRNA particles are marked by the arrows in the inset and the black dashed square represents the region of the image used for the inset (D). Scale bars: 10 μm. Inset scale bars: 2 μm. The number of cells with CHMP4B smFISH signal was then counted. The data shown in E represents the means and SD derived from three independent experiments. (F) GFP-MKLP1 expressing HeLa cells were either mock treated or treated with CEP55 siRNA for 72 h. Cells were then fixed and subjected to staining with smFISH probes against CHMP4B mRNA. The number of cells with CHMP4B smFISH signal in the MB were then counted and expressed as means and SD derived from three independent experiments. (G) HeLa cells were fixed and stained with anti-RPS6 and anti-acetylated-tubulin antibodies. Box in the image on the left indicates areas that are shown in zoomed-in images on the right. Scale bars: 10 μm. Inset scale bars: 1 μm. (H) HeLa cells expressing MKLP1-Halo (green) were fixed and stained with polyA probes (red). Arrows point to the midbody. Scale bars: 5 μm.

Validation of CHMP4B mRNA localization at the MB. (A) Midbodies were purified from HeLa cells stably expressing GFP-MKLP1. Purified midbodies were plated on poly-L-lysine coated coverslips and analyzed by fluorescence microscopy. Arrows point to GFP-MKLP1 positive midbodies. Scale bar: 10 μm. (B and C) GFP-MKLP1 expressing HeLa cells were either mock-treated or treated with CHMP4B (B) or CEP55 (C) siRNAs. Cells were then incubated for 72 h, followed by mRNA isolation and RT-qPCR analysis using CHMP4B, CEP55, and GAPDH (control) specific primers. Data shown are the CHMP4B or CEP55 mRNA levels normalized against GAPDH mRNA derived from one qPCR analysis. (D and E) GFP-MKLP1 expressing HeLa cells were either mock treated (top two panels) or treated with puromycin for 1 h (bottom two panels) and subjected to staining with smFISH probes against CHMP4B mRNA. CHMP4B mRNA particles are marked by the arrows in the inset and the black dashed square represents the region of the image used for the inset (D). Scale bars: 10 μm. Inset scale bars: 2 μm. The number of cells with CHMP4B smFISH signal was then counted. The data shown in E represents the means and SD derived from three independent experiments. (F) GFP-MKLP1 expressing HeLa cells were either mock treated or treated with CEP55 siRNA for 72 h. Cells were then fixed and subjected to staining with smFISH probes against CHMP4B mRNA. The number of cells with CHMP4B smFISH signal in the MB were then counted and expressed as means and SD derived from three independent experiments. (G) HeLa cells were fixed and stained with anti-RPS6 and anti-acetylated-tubulin antibodies. Box in the image on the left indicates areas that are shown in zoomed-in images on the right. Scale bars: 10 μm. Inset scale bars: 1 μm. (H) HeLa cells expressing MKLP1-Halo (green) were fixed and stained with polyA probes (red). Arrows point to the midbody. Scale bars: 5 μm.

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