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. 2018 Nov 8;8:16579. doi: 10.1038/s41598-018-34938-y

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Phosphoarray analysis of signaling responses to sequential treatments. (A) HCA7 cells were subjected to different regimens containing 10 μg/ml cetuximab and/or 50 μM oxaliplatin: (1) control medium for 24 hours (white), (2) control medium for 24 hours followed by oxaliplatin for 1 hour (black), (3) cetuximab for 24 hours followed by oxaliplatin for 1 hour (red), or (4) oxaliplatin for 24 hours followed by cetuximab for 1 hour (green). Phosphorylation profiles were analyzed using Proteome Profiler Human Phospho-Kinase Array Kit and quantified by densitometry. Mean +/− range is shown for four selected proteins. All results are shown in Suppl. Fig. 3. (B) HCA7 cells were subjected to different regimens containing or not 10 μg/ml cetuximab and/or 50 μM oxaliplatin as indicated in the figure. The expression or phosphorylation of selected proteins was analyzed by Western blotting. Full-length blots are presented in Supplementary Fig. 8. (C) Apoptosis of HCA7 cells was analyzed by annexin V staining after treating the cells first with or without 200 μM of the STAT3 inhibitor S31-201 for 8 hours followed by 18 hour treatment with or without 10 μg/ml cetuximab. Mean +/− SD is shown for three independent experiments. *P < 0.05.