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. 2018 Oct 31;9:4541. doi: 10.1038/s41467-018-06808-8

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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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Fig. 9 — MiR-584-5p–eIF4E3/HDAC1 regulates c-MYC and MB stem cell proliferation. a, b Western blot analysis of DAOY cells treated with miR-NC or miR-584-5p mimic (a) or scrambled or eIF4E3/HDAC1-siRNA (b) using antibodies against indicated proteins. Gel pictures are representative of three independent experiments. c Medullospheres grown from D556Med cells transfected with miR-NC or miR-584-5p mimic. Bar graphs show number of medullospheres obtained from D556Med cells transfected with miR-NC or miR-584-5p mimic. The p-value was calculated using a standard Student t-test. Error bars represent mean ± SEM of three independent experiments (performed in triplicate for each experiment). d Medullospheres grown from D556Med cells transfected with scrambled-siRNA or eIF4E3/HDAC1-siRNAs. e qPCR analysis of stem cell markers in D556Med cells transfected with miR-NC or miR-584-5p mimic. The p-value was calculated using one-way ANOVA followed by Sidak’s multiple-comparisons test. Error bars represent mean ± SEM of three independent experiments (performed in triplicate for each experiment). f qPCR analysis of MYC expression in D556Med, D425Med, D458Med, and DAOY cell lines. The p-values were calculated using standard Student t-tests. Error bars represent mean ± SEM of three independent experiments (performed in triplicate for each experiment). g Western blot analysis of c-Myc in D556Med, DAOY, D425Med, and D458Med cells transfected with miR-NC or miR-584-5p mimic. h Western blot analysis of MYC in D556Med cells transfected with siRNA against scrambled, eIF4E3, HDAC1, or c-Myc. Gel pictures in g and h are representative of three independent experiments. i miR-584-5p and eIF4E3 rescue c-Myc-dependent MB cell growth. Bar graphs show percentage of cell viability in control vector, c-Myc expression vector, or miR-584-5p mimic plus c-Myc expression vector or eIF4E3-siRNA plus c-Myc expression vector-transfected D556Med cells. Cell viability was assessed using alamarBlue. The p-value was calculated using one-way ANOVA followed by Sidak’s multiple-comparisons test. Error bars represent mean ± SEM of three independent experiments (performed in sixtuplicate for each experiment). ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05. Scale bars: 100 μM (c, d)