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. 2019 Oct 29;9:15529. doi: 10.1038/s41598-019-52112-w

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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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In vitro assay of Walker-256 cell viability and superoxide production in response to leucine treatment. Approximately 1 × 10³ W256 cells were seeded in 96-well plates and treated with 50 µM L-leucine for 24 (A and C) or 96 h (B and D). Cell viability was accessed using the neutral red uptake and the absorbance was normalised by the mean of a control group. Superoxide production was measured using dihydroethidium (DHE), and the fluorescence of each sample was normalised by the fluorescence of Hoechst 33342 (HO). Experiments were performed at least two times. Bar graphs represent the mean ± standard deviation. ***P < 0.001 versus Ctl; ^##P < 0.01 versus Leu.