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Fig. 3 | BMC Molecular and Cell Biology

Fig. 3

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

Fig. 3

RT-qPCR reveals spatial distributions alterations of mRNAs in HeLa cells and in WS fibroblast. a HeLa cells were lysed as described in the Methods and the Supplemental Information sections to obtain nuclear and cytoplasmic fractions. Anti-TRF2 antibody was used to assure that cytoplasmic extracts were not contaminated with nuclear proteins. Molecular size markers (in KiloDaltons) are shown. b Total RNA was extracted from each fraction and analyzed by RT-qPCR using the indicated set of primers (Supplementary Table 2). The results of nuclear/cytoplasmic ratio were plotted using GraphPad Prism. c WS patient-derived fibroblasts and the derivative cell line with ectopic expression of flag-WRN (WS/F-WRN) were lysed as in (a) to obtain nuclear and cytoplasmic fractions. Molecular size markers (in KiloDaltons) are shown. d Total RNA was extracted from each fraction and analyzed by RT-qPCR using primers sets as in (b) and the results were plotted using GraphPad Prism. e Cells were first incubated in media containing dox for three days. At day three, the cells were washed gently and incubated for 3 additional days in media without dox. Next, the cells were lysed, and extract were processed as describe in (a). Western blot analysis was used to assess WRN expression. Molecular size markers (in KiloDaltons) are shown. f The nuclear and cytoplasmic fractions were used to isolated total RNA, which was analyzed by RT-qPCR with primers for the indicated transcripts. In (b) and (f) a Two-way ANOVA followed by Sidak’s multiple comparisons test was used to calculate only the significance between shCTR and shWRN HeLa cells after dox induction. The error bars represent the mean ± SEM (n = 3). * p value < 0.05; ** p value < 0.001; *** p value ≤0.0002; ns, no significant differences. In (d) the experiment was performed in two biological replicates, thus the means and ± SEM without statistical analysis are shown

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