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. 2019 Mar 21;9:5012. doi: 10.1038/s41598-019-41380-1

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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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Enhanced cytotoxicity of TMZ + RSV in vitro and in vivo is mediated by increased autophagy. (A) Immunoblotting of apoptotic and autophagic marker proteins in control and drug treated C6 cells. Cells were treated with indicated drug(s) concentrations for 72 h and whole cell lysates were prepared. Protein expression levels of Caspase 3, LC3B, Beclin1, pCdk5 and beta actin were assessed by immunoblotting. (B) Immunohistochemical staining for autophagy marker, LC3B in tumor sections from vehicle, TMZ, RSV and TMZ + RSV treated rats. (C) Effect of 3-MA, an early stage autophagy inhibitor on the cytotoxicity of various drug treatments as indicated in the figure. (D) Effect of Caspase 3 inhibitor (Z-DEVD FMK) on the cytotoxicity of various drug treatments as indicated in the figure. For C and D, C6 cells were pretreated with indicated concentrations of inhibitors for 2 h and then treated with TMZ, RSV or TMZ + RSV for 72 h. This was followed MTT assay to determine percent cell survival. *p < 0.05, **p < 0.005.