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. 2020 Nov 18;12:178. doi: 10.1186/s13148-020-00964-9

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© The Author(s) 2020

Open AccessThis 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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 2 — The combined effects of panobinostat and arsenic trioxide (ATO) include the differentiation of A/E9a;Nras ^G12D-leukemic blasts by panobinostat and induction of apoptosis by ATO. (A) Flow cytometry analysis of the cell surface expression of c-Kit; Sca1; Mac1 and Gr1 of GFP-positive tumor cells in the bone marrow of A/E9a;Nras ^G12D tumor-bearing mice treated for 5 days with either panobinostat (pan; 25 mg/kg) or panobinostat combined with ATO (2.5 mg/kg). (n = 3; data are expressed as mean plus SEM; *P <0.05, **P <0.001) (B) Representative dot plots of AnnexinV-PI staining of tumor cells isolated from the bone marrow of A/E9a; Nras^G12D tumor-bearing mice treated for 4 hours with either panobinostat (25 mg/kg) or ATO (2.5 mg/kg) or a combination. Numbers given are the percentage of total cell population. (C) Normalized expression of AnnexinV on tumor cells treated with vehicle, panobinostat, ATO or a combination (n = 3; data are expressed as mean plus SEM). (D) Quantification of terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) positivity as a proportion of total cell area (n = 3; *P <0.001) (E) Representative images of hematoxylin-eosin staining (H&E; upper panels) and analysis of apoptotic cells by TUNEL staining and counterstained with hematoxylin (lower panels). Staining was performed on de-calcified femurs isolated from A/E9a;Nras ^G12D tumor-bearing mice treated for 4 hours as indicated. Imaging was performed using 60x objective (scale bars = 50 μm).