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Fig. 2 | BMC Molecular and Cell Biology

Fig. 2

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

Fig. 2

WRN depletion does not affect the levels of ribosomal proteins or ribosomal rRNAs. a A representative Western blot of WRN depletion after 3 days of dox treatment using actin as loading control. b Representative Western blot of nuclear-cytoplasmic fractionation of HeLa cells. The absence of cross contamination between nuclear and cytoplasmic fractions was confirmed using Lamin A/C (nuclear marker) and Tubulin (cytoplasmic marker). WRN depletion was confirmed in the same blot. Molecular size markers (in KiloDaltons) are shown. c Total RNA was purified from the Polysome fractions as described in the Methods and the Supplemental Information sections, and used for quantitative RT-qPCR analysis with sets of primers against 5.8S, 18S and 28S rRNAs. GAPDH was used as the internal control. The significance of the fold changes in three biological replicates was calculated using two-way ANOVA followed by Sidak’s multiple comparisons test from GraphPad Prism. The error bars represent the mean ± SEM (n = 3). ns, no significant difference. d A representative Western blot analysis of whole cell lysates derived from shCTR control and WRN-depleted cells 72 h after dox induction using antibodies against the indicated ribosomal proteins. Molecular size markers (in KiloDaltons) are shown for both experiments. e Band intensity determination in three biological replicates was generated using ImageJ (NIH) and plotted using GraphPad Prism. f Quantitative RT-qPCR analysis of ribosomal proteins genes using GAPDH as the internal control was performed as previously described, and the results of three biological replicates were plotted using GraphPad Prism. e and f, Two-way ANOVA followed by Sidak’s multiple comparisons test from GraphPad Prism, was used to calculate the significance in both approaches. The error bars represent the mean ± SEM (n = 3). ** p value < 0.01; ns, no significant differences

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