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. 2019 Feb 20;9:2405. doi: 10.1038/s41598-019-39555-x

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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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Convallatoxin modulate MOR endocytosis independently of Na⁺/K⁺-ATPase. (A) High-throughput screen to identify enhancers of MOR endocytosis. Results of the primary screen showing significant MOR endocytosis in convallatoxin + morphine treatment. Data are presented as percentages of the values for morphine (0.3 μM; ~EC₁₀) alone. (B) Chemical structure of convallatoxin. (C) Concentration-response curves for morphine and methadone induction of MOR endocytosis. (D) Concentration-response curves for morphine induction of MOR endocytosis in the presence or absence of convallatoxin. (E) Concentration-response curves of convallatoxin in morphine mediated MOR endocytosis. (F) The effects of cardiac glycosides on MOR endocytosis are determined by treating U2OS-MOR cells with various concentrations of gitoxigenin, bufalin, and convallatoxin. Chemical structure of bufalin, and gitoxigenin. (G,H) Involvement of Na⁺/K⁺-ATPase in the effect of cardiac glycosides. U2OS-MOR cells were transiently transfected with sh-control (G) or sh-Na⁺/K⁺-ATPase α1 (H) for 24 h prior to MOR internalization assay. (I) Concentration-response curves for morphine-induced MOR endocytosis with or without rostafuroxin. (J) Concentration-response curves of rostafuroxin in morphine-mediated MOR endocytosis. RLU, relative light units. Data in C-J, MOR internalization was measured by an enzyme complementation assay in U2OS-MOR cells. All values indicate the mean ± SD.