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. 2018 Feb 14;9(2):220. doi: 10.1038/s41419-018-0295-5

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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. 1 — a Genes in glutaminolysis pathway were upregulated in MYCN-amplified tumors (93 cases) compared with non-MYCN-amplified tumors (550 cases) (ANOVA, p < 0.01). These data were derived from Kocak dataset (GSE45547). Age (red > 18 months; green < 18 months); MYCN (red = amplification; green = non-amplification); stage (red = stage 4; blue = stage 4S; brown = stage 3; dark green = stage 2, green = stage 1). b MYCN protein and mRNA expression levels were determined by immunoblot (left panel) and qPCR (right panel), respectively. NB-1643 control-shRNA-inducible and MYCN-shRNA-inducible cells were collected after 3 days in the absence or presence of 1 μg/ml doxycycline for MYCN level analysis. mRNA levels in cells expressing control-shRNA were set to 1. Error bars represent standard deviation from the mean of triplicate samples. Data are representative of two independent experiments. c Glycolysis and glutaminolysis as determined by the generation of ³H₂O from [5-³H]glucose and the generation of ¹⁴CO₂ from [U-¹⁴C]glutamine, respectively. NB-1643 control-shRNA and MYCN-shRNA cells (collected after incubation with 1 μg/ml doxycycline for the expression of specific RNA in 2% FBS media for 3 days, followed by overnight 10% FBS media recovery) were used for measuring the metabolic flux. Error bars represent standard deviation from the mean of triplicate samples. Data are representative of two independent experiments. d mRNA levels of genes essential for glutaminolysis in NB-1643 cells were determined by qPCR. NB-1643 control-shRNA-inducible and MYCN-shRNA-inducible cell lines were treated with 1 μg/ml doxycycline for 3 days before harvesting the samples for RNA extraction followed by qPCR analyses of metabolic genes in the glutaminolytic pathway. mRNA levels in NB-1643 control-shRNA cells were set to 1. Error bars represent standard deviation from the mean of triplicate samples. Data are representative of two independent experiments. e Chromatin immunoprecipitation assessment of the binding of MYCN on indicated genes after 24-h MYCN induction with 500 ng/ml doxycycline in MycN3 cells. MYCN was immunoprecipitated from cross-linked DNA, and then DNA was extracted for PCR with primers that amplify predicted binding site and non-binding site for each gene