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. 2019 Oct 7;9:14394. doi: 10.1038/s41598-019-50840-7

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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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Inhibition of HSPAs by VER and JG-98 lead to apoptosis in NCI-H23 but not in NCI-H1299 cells. (A) Cell death detection after 72 h treatment of NCI-H23 and NCI-H1299 cells with VER-155008 (VER). Results of propidium iodide (PI) uptake test show mean values ± SD (n = 3, each in duplicate). Statistical significance was determined using two-tailed t-test. (B,C) Flow cytometry analysis of cell death in NCI-H23 cells incubated with VER performed using Annexin V (AV)/PI double labeling method. Cells were plated onto 6-well dishes (2 × 10⁴/well) and treated for 48 h. Graph shows percentage of cells labeled with AV and/or PI (mean value ± SD, n = 3). (D,E) Distribution of the cell cycle phases in NCI-H23 (D) and NCI-H1299 (E) cells non-treated (0) or treated for 48 h with VER (5, 10, 20). Cell cycle phases distribution was analyzed immediately after treatment (72 h T) or followed by 48 h of recovery (48 T + 48 R). Graph shows percentage of cells (mean ± SD) calculated from seven (NCI-H1299 cells) or two (NCI-H23) independent experiments, each in duplicate. Statistical significance was calculated using two-tailed t-test. (F) Cell death detection after 72 h treatment of NCI-H23 and NCI-H1299 cells with JG-98. Results of trypan blue staining assay show mean values ± SD (n = 3, each in duplicate). (G) Western blot analysis of activated caspase-3 and cleaved PARP in cells non-treated and treated with JG-98 or VER (72 h). Representative immunoblots are shown (n = 2), actin was used as a protein loading control. In (A), (C) and (F) statistical significance was determined by one-way analysis of variance (ANOVA) and Scheffe’s pairwise post hoc test.