Figure 6.
The 3′ UTR of CHMP4B is important for protein accumulation and mRNA translocation. (A) HeLa cells expressing various RFP-tagged dox-inducible constructs were incubated with 2 μg/ml doxycycline for 48 h and then subjected to immunostaining with anti-acetylated-tubulin. The asterisks mark the MB and the white dashed square represents the region of the image used for the inset. Scale bars: 10 μm. Inset scale bars: 5 μm. (B and C) Ratio between RFP fluorescence in the intercellular bridge and cell body was calculated from 60 randomly chosen cells in telophase. B shows distributions derived from individual cells (each dot represents a single cell). Statistical analysis in C was done on the means, and SD were derived from the three independent experiments where experimental means were calculated by averaging values from all the cells from each experiment. Each experiment is represented by a different shade of color. Statistical analysis is represented with a P value. (D) HeLa cells expressing various RFP-tagged dox-inducible constructs were incubated with 2 μg/ml doxycycline for 48 h and then subjected to immunostaining with anti-acetylated-tubulin. The percentage of cells with multinucleation was then counted. Shown data are the means and SD derived from three independent experiments. Statistical analysis is represented with a P value. (E) HeLa cells expressing various RFP-tagged dox-inducible constructs were incubated with 2 μg/ml doxycycline for 48 h and then subjected to immunostaining with anti-acetylated-tubulin. The percentage of cells in telophase was then counted. Shown data are the means and SD derived from three independent experiments. Statistical analysis is represented with a P value. (F) HeLa cells expressing dox-inducible RFP-CHMP4B or RFP-CHMP4B-3′UTR constructs were incubated with 2 μg/ml doxycycline for 48 h and subjected to staining with smFISH probes against CHMP4B. The cell outline is marked in yellow with the asterisks marking the MB. The black dashed square represents the region of the image used for the inset. Scale bars: 10 μm. Inset scale bars: 2 μm. (G) Whole-cell or MB-associated mRNA was isolated from these cell lines expressing various RFP-tagged constructs and subject to RT-qPCR analysis using RFP or GAPDH (control) specific primers. RFP mRNA levels were then normalized against GAPDH mRNA and expressed as a ratio between MB and whole-cell RFP mRNA levels. Statistical analysis is represented with a P value and was done on the means and SD derived from three independent experiments.

The 3′ UTR of CHMP4B is important for protein accumulation and mRNA translocation. (A) HeLa cells expressing various RFP-tagged dox-inducible constructs were incubated with 2 μg/ml doxycycline for 48 h and then subjected to immunostaining with anti-acetylated-tubulin. The asterisks mark the MB and the white dashed square represents the region of the image used for the inset. Scale bars: 10 μm. Inset scale bars: 5 μm. (B and C) Ratio between RFP fluorescence in the intercellular bridge and cell body was calculated from 60 randomly chosen cells in telophase. B shows distributions derived from individual cells (each dot represents a single cell). Statistical analysis in C was done on the means, and SD were derived from the three independent experiments where experimental means were calculated by averaging values from all the cells from each experiment. Each experiment is represented by a different shade of color. Statistical analysis is represented with a P value. (D) HeLa cells expressing various RFP-tagged dox-inducible constructs were incubated with 2 μg/ml doxycycline for 48 h and then subjected to immunostaining with anti-acetylated-tubulin. The percentage of cells with multinucleation was then counted. Shown data are the means and SD derived from three independent experiments. Statistical analysis is represented with a P value. (E) HeLa cells expressing various RFP-tagged dox-inducible constructs were incubated with 2 μg/ml doxycycline for 48 h and then subjected to immunostaining with anti-acetylated-tubulin. The percentage of cells in telophase was then counted. Shown data are the means and SD derived from three independent experiments. Statistical analysis is represented with a P value. (F) HeLa cells expressing dox-inducible RFP-CHMP4B or RFP-CHMP4B-3′UTR constructs were incubated with 2 μg/ml doxycycline for 48 h and subjected to staining with smFISH probes against CHMP4B. The cell outline is marked in yellow with the asterisks marking the MB. The black dashed square represents the region of the image used for the inset. Scale bars: 10 μm. Inset scale bars: 2 μm. (G) Whole-cell or MB-associated mRNA was isolated from these cell lines expressing various RFP-tagged constructs and subject to RT-qPCR analysis using RFP or GAPDH (control) specific primers. RFP mRNA levels were then normalized against GAPDH mRNA and expressed as a ratio between MB and whole-cell RFP mRNA levels. Statistical analysis is represented with a P value and was done on the means and SD derived from three independent experiments.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal