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. 2019 Feb 28;9:3015. doi: 10.1038/s41598-019-39418-5

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© The Author(s) 2019

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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ELT3-245 cells exhibit enhanced anchorage-independent cell growth and resistance to anoikis. (A) Automatic particle analysis of colonies from soft agar assay. ELT3-245 cells formed significantly bigger colonies, compared to ELT3 cells (****P < 0.0001). (B) 24 hours after plating ELT3 and ELT3-245 cells in soft agar, cultures were continuously treated with 20 nM rapamycin for 3 weeks. Rapamycin treatment did not cause a significant reduction in colony volume in ELT3-245 cells, compared to DMSO. However, rapamycin caused a significant increase in colony volume in ELT3 cells (*P = 0.0148). (C) A subset of ELT3 cells are rapamycin-resistant. Volume distribution of DMSO- and rapamycin-treated parental ELT3 in soft agar. Approximately 19% of rapamycin-treated ELT3 colonies were bigger than DMSO-treated ELT3 colonies. (D) The top 19% of rapamycin-treated ELT3 colonies were significantly bigger, compared to the bottom 81% of rapamycin-treated ELT3 colonies (P < 0.0001). The top 19% of rapamycin-treated ELT3 colonies were not significantly bigger, compared to DMSO-treated ELT3-245 colonies (P > 0.05). (E) ELT3 and ELT3-245 cells were grown in non-adherent condition for 6 and 24 hours, harvested and the percentage of dead cells was counted by trypan blue exclusion. 0 h indicates percentage of dead cells immediately after trypsinization and resuspension in growth media. (F) Immunoblotting of lysates from cells grown in suspension conditions for 0, 6 and 24 hours from panel E. Full-length blots are presented in Supplementary Fig. 6.