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. 2017 Sep 11;45(20):11725–11742. doi: 10.1093/nar/gkx782

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© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 6. — Silencing NuMA and treatments with actinomycin D and H₂O₂ lead to p27^kip1 increase unless it is already elevated. MCF10A WT-p53 cells and p53-null MDA-MB-157 cells were treated with either siRNA against NuMA (siNuMA) or non-targeting siRNA (siNT) and left in culture for 10 (MCF10A) or eight (MDA-MB-157) days, and/or with 0.08 μg/ml actinomycin D (Act D) or control DMSO for 4 h on day 10 (or 8), and/or with 250 μM of H₂O₂ for 4 h on day 10 (or 8). (A and C). Percentage of cells with perinucleolar caps of fibrillarin indicative of nucleolar stress (n = 3; 150 nuclei analyzed per condition) in MCF10A (A) and MDA-MB-157 (C) populations. (B and D). Levels of FUrd staining for nascent rRNA (nucleolus) following normalization to nucleoplasmic staining intensity (nascent mRNAs) under the different treatment conditions (n = 3; 100 nuclei analyzed per condition). Shown is a representative western blot of NuMA expression upon siNuMA treatment in MCF10A cells (B) and MDA-MB-157 cells (D). (E and F). Western blot and accompanying quantification (following standardization with lamin B and compared to the first band of each western blot) for p27^kip1, following Act D or H₂O₂ treatment in the presence (siNuMA) or absence (siNT) of siRNA against NuMA; lamin B is used as loading control in MCF10A (E) and MDA-MB-157 (F) cells. (G). Western blot for p53, NuMA, p27^kip1 and loading control lamin B in untreated S1, MCF10A and MDA-MB-157 cells.