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. 2017 Jan 16;36(23):3274–3286. doi: 10.1038/onc.2016.472

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Copyright © 2017 The Author(s)

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/

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Effects of Nutlin-3 on IGF-1R modulated biological activities. (a) Effect of Nutlin-3 on growth in soft agar. BE and DFB cells were plated into 10% serum containing soft agar and treated with dimethyl sulfoxide (DMSO) or 1 μM Nutlin-3 for 14 days. The colonies were counted and the results from three independent experiments are displayed as mean±s.e.m. relative to untreated control (C). (b) Effect of Nutlin-3 on cell viability in suspension. Cells plated into poly-2-HEMA-coated wells in serum-free medium were treated without and with 1 μM Nutlin-3 for 48 h in the presence or absence of IGF-1. The cell viability was measured by PrestoBlue reagent and displayed as a percentage of untreated controls. Data correspond to the mean±s.e.m. from three independent experiments. (c and d) Effect of Nutlin-3 on cell cycle distribution and IGF-1-dependent cell viability. Cells growing in monolayer, treated as indicated, were analyzed for cell cycle distribution (G1, S and G2/M) (c) or cell viability (d). Results from three independent experiments are displayed as mean±s.e.m., percentage of total cell population (c) or as mean±s.e.m. relative to untreated cells (d). The levels of Nutlin-3-mediated IGF-1R degradation at 48 h were quantified in parallel samples by WB using GAPDH as a loading control (e). Statistical analysis: Nutlin-3-treated cells compared with control-treated cells: *P<0.05, ***P<0.001.