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. 2018 Jun 5;46(12):6188–6205. doi: 10.1093/nar/gky455

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© The Author(s) 2018. 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/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 5. — Identification of a role for p14ARF in TIF-IA degradation. (A) TIF-IA interacts with p14ARF in response to aspirin. SW480 cells were treated with 0–5 mM aspirin for 16 h. Immunoprecipitation was carried out on WCL using antibodies to TIF-IA and IgG control. Precipitated proteins were subjected to western blot analysis with the indicated antibodies. Input levels are shown. (B–F) Silencing of P14ARF is required for stress-mediated degradation of TIF-IA. (B, C, E and F) SW480 cells were transfected with control or p14ARF siRNA (siARF) then treated for 16 h with CDK4i (0–1 μM) or aspirin (0–3 mM). (B) Immunoblot analysis was performed on WCL with the indicated antibodies. (C) TIF-IA levels relative to actin were quantified using ImageJ analysis. Graph shows the mean (±s.e.m.) compared to non-treated, siControl. N = 3. (E and F) Immunomicrographs (×63) show the levels and localisation of TIF-IA in fixed cells. (E) IPlab software quantified nuclear (as depicted by DAPI staining) intensity of TIF-IA. Data are the mean (±s.e.m.) of >150 nuclei. N = 3. Inset shows nucleolar (outlined) TIF-A in p14ARF transfected cells treated with aspirin. (D) SW480 cells were transfected with pcDNA3 control plasmid (–) or pcDNA3-p14ARF (+) then either non-treated or treated with aspirin (3 mM, 16 h). Immmunoblot was performed on WCL with the indicated antibodies. Actin acts as a loading control throughout. Scale bar = 10 μm. N values are biological repeats. P values were derived using a two-tailed Student's t test. See also supporting Supplemental Figure S4.