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. 2014 Oct 30;13(18):2931–2943. doi: 10.4161/15384101.2014.946869

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© 2014 The Author(s). Published with license by Taylor & Francis Group, LLC

© Birgit Steinmetz, Hubert Hackl, Eva Slabáková, Ilse Schwarzinger, Monika Smějová, Andreas Spittler, Itziar Arbesu, Medhat Shehata, Karel Souček, and Rotraud Wieser

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 7. — Experimental down-regulation of GDF15 counteracts EVI1 enhancement of the ATRA induced cell cycle arrest. (A) U937_vec and U937_EVI1 cells infected with a control shRNA targeting Renilla luciferase (shRen) or shRNAs against GDF15 (shGDF15-1, shGDF15-2) were treated with solvent or ATRA for 24 h, and GDF15 mRNA levels were measured by qRT-PCR. GDF15 expression was normalized to that of B2M using the ΔΔ^Ct method⁸⁸ and ATRA treated U937_EVI1_shRen cells as a calibrator. Results represent means + SEs from 3 independent experiments. (B) U937_vec and U937_EVI1 cells containing shRen, shGDF15-1, or shGDF15-2 were treated with solvent or ATRA for 5 days, and the GDF15 precursor was detected by immunoblot analysis. α-tubulin was used as a loading control. (C) U937_vec and U937_EVI1 cells containing shRen, shGDF15-1, or shGDF15-2 were treated with solvent or ATRA for 5 days, and mature GDF15 was detected in culture supernatants by ELISA. Results represent means + SEs from 5 independent experiments. Due to variation in factors results were not significant, but the enhancement of the ATRA induction by EVI1 in shRen infected cells, as well as the knockdown of GDF15 by both shRNAs were consistently seen in all experiments. (D) U937_vec and U937_EVI1 cells containing shRen, shGDF15-1, or shGDF15-2 were treated with solvent or ATRA for 5 d and subjected to cell cycle analysis by propidium iodide staining and flow cytometry. Results represent means + SEs from 7 independent experiments. Significance was calculated using Student's 2-tailed t-test (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant).