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. 2019 Jul 9;9:9895. doi: 10.1038/s41598-019-46163-2

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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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Reversine induces DNA damage and apoptosis markers in JAK2^V617F-positive cell lines. (A) Western blot analysis for AURKA, AURKB, phospho(p)-histone H3^S10, p-histone H2A.X^S139 (γ-H2A.X) and Caspase 3 (total and cleaved) in total cell extracts from SET2 and HEL cells treated with graded concentrations of reversine (vehicle, 1, 2.5, 5, 10 or 25 μM) for 48 hours; membranes were reprobed with the antibody for the detection of the respective total protein or α-tubulin, and developed with the SuperSignal™ West Dura Extended Duration Substrate system using a Gel Doc XR+ imaging system. Cropped blots are shown and full-length blots are included in Supplementary Fig. 7. (B) Immunofluorescence analysis of SET2 and HEL cells treated or not with 5 μM reversine for 24 hours, displaying α-tubulin (green) and DAPI (blue) staining. Blank and red arrows illustrate mitotic catastrophe and nuclear fragmentation, respectively. Scale bars are shown in the figure (50 μm). Bar graphs represent the quantification of morphological findings of at least 200 cells for each condition. (C) Gene expression heatmap from qPCR array analysis of SET2 cells treated with reversine (5 μM). The mRNA levels are normalized to those of vehicle-treated SET2 cells and calculated as fold change in expression; genes demonstrating ≥1.5-fold in either direction compared to vehicle-treated cells upon the treatment with reversine are included in the heatmap. Two independent experiments of each condition were used for the analysis; green indicates repressed mRNA levels and red elevated mRNA levels. (D) qPCR analysis of BCL2, BCL2L1, BIRC5, BNIP3, BNIP3L, and BIK mRNA expression in SET2 and HEL cells treated with reversine (5 μM). The dashed line represents the mean gene expression in vehicle-treated cells and bars represent the fold change in gene expression in SET2 and HEL cells treated with reversine (5 μM) compared to their respective untreated cells. The p values and cell lines are indicated in the graphs. **p < 0.01, **p < 0.001; Student t test.