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. 2018 Nov 15;8:16837. doi: 10.1038/s41598-018-35198-6

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© The Author(s) 2018

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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Avocatin B increases ATF4 activation and AMPK-mTOR signaling in AMfL cells co-cultured with BM adipocytes. (A) THP-1 cells were co-cultured with BM adipocytes for 24 hours with or without avocatin B (10 μM), and expression levels of ATF4 protein were detected by immunoblotting; Cont, controls. Results shown are representative of three independent experiments. (B) OCI-AML3 cells transfected with control short hairpin RNA (shC) or shRNA against AMPK (shAMPK) were treated with the indicated concentrations of avocatin B for 48 hours in the presence or absence of BM adipocytes under serum-starved conditions. The effects on cell viability were determined by cell counts using the trypan blue exclusion method. Graphs show the mean ± SD of the results from three independent experiments. (C) OCI-AML3 cells transfected with control short hairpin RNA (shC) or shRNA against AMPK (shAMPK) were cultured with or without avocatin B (10 μM) and AraC (3 μM) for 18 hours in the presence or absence of BM adipocytes. Expression levels of AMPK, p-AMPK, 4E-BP1, p-4E-BP1, S6, p-S6 and α-tubulin proteins in the cells were detected by immunoblotting; Cont, controls. Results shown are representative of three independent experiments. (D) OCI-AML3 cells transfected with control short hairpin RNA (shC) or shRNA against AMPK (shAMPK) were cultured with or without avocatin B (20 μM) in the presence or absence of BM adipocytes under serum-starved conditions and glucose uptake measured. Plated cells were treated with avocatin B for 2 hours. Fluorescent signal was measured with a plate reader using the bottom read mode. Graphs show the mean ± SD of the results from three independent experiments. *p < 0.05. (E) OCI-AML3 cells transfected with control short hairpin RNA (shC) or shRNA against AMPK (shAMPK) were cultured with or without avocatin B (20 μM) for 2 hours in the presence or absence of BM adipocytes under serum-starved conditions. Cells were plated at 50,000 cells/well, after which fatty acid (FA) uptake was determined by adding a fatty acid mixture (dodecanoic acid fluorescent fatty acid substrate) and incubating for 1 hour. Fluorescent signal was measured with a plate reader using the bottom read mode. Graphs show the mean ± SD of the results from three independent experiments. **p < 0.01; *p < 0.05.