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Fig. 4 | BMC Molecular and Cell Biology

Fig. 4

From: WRN modulates translation by influencing nuclear mRNA export in HeLa cancer cells

Fig. 4

FISH analysis shows a significative change in mRNA distribution after WRN depletion in HeLa cells. a Representative Western blot showing WRN levels in shCTR and shWRN HeLa cells after 72 h of dox treatment. Molecular size markers (in KiloDaltons) are shown. b shCTR and shWRN HeLa cells were fixed and hybridized with Oligo (dT)50-Cy3 as described in Supplemental Information. Representative images showing the mRNA signal in each cell line (Oligo (dT)50-Cy3) and the yellow line drawn across cells used to measure the fluorescence intensity in pixels (Scale bar = 50 μm). c The nuclear periphery (as defined by DAPI staining) was used to separate the intensities of the cytoplasmic and nuclear Poly (A)+ signal and the obtained data were plotted using ImageJ. The sum of the resulting intensities from the nuclei and cytoplasm quantitation were used to calculate the nuclear/cytoplasm mRNA ratio in shCTR and shWRN cell and plotted using GraphPad Prism (bottom graph). d For each cell analyzed, the average of the fluorescence intensity (pixels) in the nuclei was divided by the average of the fluorescence intensity (pixels) of the cytoplasm and the resulting data was used to generate the graph. Two-tailed unpaired t test from GraphPad Prism was used to calculate the significance between WRN-depleted and control cells. The error bars represent the mean ± SEM of three independent experiments (shCTR n = 342 cells, shWRN n = 389). **** p value < 0.0001

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