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. 2018 Mar 2;8:3930. doi: 10.1038/s41598-018-22250-8

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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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Cell signal mechanism of metformin pretreatment in terms of enhancement of T-DM1 efficacy. (A) The western blot results demonstrated that when BT-474 cells were pretreated with metformin and then treated with metformin in combination with T-DM1, the levels of phosphor-mTOR, AKT and MAPK were decreased. The original full-length western blot images are showed in Supplementary Figure S4. (B,C) Cells were pre-treated with caveolin-1 shRNA to create caveolin-1-deficient cells. Then, cells were treated with metformin followed by T-DM1 and metformin combined treatment. The data showed that in caveolin-1 knockdown cells, p-AKT and Bcl2 were not decreased, while p-MAPK was slightly affected. The original full-length western blot images are showed in Supplementary Figure S5. (D) Cells prepared as in (C) were subjected to MTT assay. In comparison with the mock group, which also underwent M/MT treatment, the cytotoxic effect was significantly reduced in caveolin-1 knockdown cells. shCAV-1: caveolin-1 shRNA; Ctr: control; M: metformin; T: T-DM1. Value = mean ± SD, *P < 0.05. The western blots were derived under the same experimental conditions from the same cell lysates of each treatment group in (A–C).