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. 2018 Aug 27;8:12920. doi: 10.1038/s41598-018-31332-6

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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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MDR mechanism in ALI multilayered mono-cultures: (A) Schematics of MDR in cancer cells triggered by overexpression of MRP1/ABCC1 and MDR1/ABCB1 drug efflux pumps. (B) Western blot analysis of the expression of MRP1/ABCC1 and MDR1/ABCB1 drug efflux pumps in A549 cells forming ALI multilayered mono-cultures grown for 14 d and then exposed to docetaxel (Doc), vinblastine (Vin), cytarabine (Cyt) or methotrexate (Met) at their nominal IC₅₀ concentration for 72 h. Cell cultures were exposed to drugs by direct inoculation. The expression of these pumps in untreated cultures (NT) is also reported for comparison. Abbreviations “n₁”, “n₂” and “n₃” indicate different experimental replicates. β-actin expression is reported as proteins loading control. (C) Schematics of the mechanism of action of reversan, a selective MRP1/ABCC1 inhibitor. (D) Histogram of the LDH activity in the experimental controls: untreated ALI multilayered mono-cultures (NT), cell-free supplemented cell medium (DMEM), ALI multilayered mono-cultures exposed to reversan (10 μM) for 72 h, and positive control (LDH PT). No significant LDH activity was detected following reversan treatment. Data are reported as average ± standard error of the mean (n_replicates = 2; n_tests = 3). p < 0.01 indicates a significant difference from NT (one-way ANOVA with Dunnett post-test). (F) Percentage (%) cytotoxicity detected by LDH cytotoxicity assay in ALI multilayered mono-cultures grown for 14 d and exposed to 10 concentrations of docetaxel for 72 h, in the presence or absence of reversan (10 μM). Cell cultures were exposed to drugs by direct inoculation. Values for untreated cultures (NT) and positive control (LDH PT) are also shown. Data are reported as average ± standard error of the mean (n_replicates = 2; n_tests = 3). Differences were not significant (two-way ANOVA with Bonferroni post-test).