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. 2020 May 14;11:2393. doi: 10.1038/s41467-020-16212-w

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© The Author(s) 2020

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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Fig. 4 — a Representative images for interphase and metaphase FISH analysis for EML4-ALK fusion and amplification status. Separation of 3′ (red) probe from 5′ (green) probe indicates ALK fusion event (orange arrows). The scale bars represent 5 μm. b Frequency of cells with the indicated EML4-ALK fusion and amplification status in the gradually evolved erALK-TKI cell lines (lines 0 were analyzed). c Impact of CRISPR-mediated genetic ablation of ALK on clonogenic survival of the indicated H3122 derivates. Mean ± SD of experimental duplicates, representing separate dishes with alternative ALK directed guide RNAs; representative colonies are shown. The scale bars represent 100 μm. d Evaluation of EML-ALK ablation by immunoblotting analysis. Raw images shown in Supplementary Fig. 14. e Immunoblot evaluation of the expression and activity of EML4-ALK oncogenic signaling in the presence of Crizotinib or after 48 h of drug holidays, for the indicated cell lines with evolved and engineered resistance. ALK o/e and ALK o/e' denote independently derived sublines. Raw images shown in Supplementary Fig. 15. f Impact of retrovirally mediated overexpression of EML4-ALK fusion and its L1196M mutant variant on sensitivity to crizotinib, measured by Cell Titer Glo assay. Mean ± SD of experimental triplicates representing separate wells are shown.