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. 2016 Sep 9;15(21):2958–2972. doi: 10.1080/15384101.2016.1231281

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© 2016 The Author(s). Published with license by Taylor & Francis

This is an Open Access article distributed under the terms of the Creative Commons Attribution-Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The moral rights of the named author(s) have been asserted.

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Figure 5. — Post-transcriptional and Post-translational p53-dependend Pathways Cooperate to Down-regulate CDC7 in Cells Undergoing DNA Damage. (A) Immunoblotting analysis of whole cell extracts (WCE) from IMR90 cells treated with either p53 siRNA, miR-192/215 siRNA, Fbxw7β shRNA, a combination of miR-192/215 siRNA and Fbxw7β shRNA, GSK3β siRNA or Cdh1 siRNA. β-actin was used as a loading control. (B) DNA content distribution analysis of indicated samples as described in (A) is presented as a bar diagram. Values above the bars represent the percentage of cells with S-phase DNA content. (B) Representative of 3 independent experiments. (C) Residual p53-, Fbxw7β-, miR-192/215- and GSK3β mRNA as measured by qRT-PCR in percentages (%) relative to the respective control (CTRL). (C) Mean ± SEM from 3 independent experiments (D) 2 μM Doxorubicin (Doxo) was added to IMR90 cells for 48h, after 24h pre-treatment with either p53 siRNA, miR-192/215 siRNA, Fbxw7β shRNA, a combination of miR-192/215 siRNA and Fbxw7β shRNA or GSK3β siRNA. CTRL represents the combination of non-targeting control siRNA and DMSO treatment. (E) DNA content distribution analysis of indicated samples as described in (D) is presented as a bar diagram. Values above the bars represent the percentage of cells with S-phase DNA content. (E) Representative of 3 independent experiments. (F) 1 nM Actinomycin D (ActD) was added to HCT116 cells for 24h, after 24h pre-treatment with following siRNAs: either p53 siRNA, miR-192/215 siRNA, Fbxw7β shRNA, a combination of miR-192/215 siRNA and Fbxw7β shRNA or GSK3β siRNA. CTRL represents the combination of non-targeting control siRNA and DMSO treatment. (G) DNA content distribution analysis of indicated samples as described in (F) is presented as a bar diagram. Values above the bars represent the percentage of cells with S-phase DNA content. (G) Representative of 3 independent experiments.