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. 2017 Sep 25;7:12283. doi: 10.1038/s41598-017-12060-9

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© The Author(s) 2017

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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Effect of neferine and DOX-treatment on DNA breaks, intrinsic apoptosis and morphological changes in H9c2 cells (A) Terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling showing reduced DNA breaks and apoptotic body formation by neferine pre-treatment. (B) % of TUNEL positive apoptotic nuclei. (C) Western blot analysis of neferine and DOX-treatment on pro- and anti-apoptotic protein levels. (D) Densitometry analysis of the protein bands of pro- and anti-apoptotic proteins. (E) Phase-contrast microscopic images of H9c2 cells. (i) Control cells. (ii) Neferine treated cells showing normal morphology. (iii) DOX-alone treated cells showing spindle shape shrinkage and detachment of cells from culture surface. (iv) Cells pre-treated with neferine followed by DOX-treatment reduced the shrinkage and rounding up of cells from the culture surface. (F) Morphological analysis of H9c2 cells by scanning electron microscopy. (i & ii) Control and neferine treatment showing the normal morphological architectures of elongated spindle shaped H9c2 cells with dense surface. (iii-v) DOX-treatment showing morphological changes like loss of spindle shape, cell shrinkage, rounding up of cells and membrane blebbing associated with apoptosis in H9c2 cells. (vi-viii) Neferine pre-treatment showing reduced structural abnormalities induced by DOX- treatment. The graphs represent mean ± s.d. of three individual experiments. ^*p < 0.05 significantly different from control, ⁺p < 0.05 significantly different from DOX treated cells, n.s. = non-significantly different from control (one way ANOVA followed by Tukey’s multiple comparison).