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. 2017 Jul 26;7:6516. doi: 10.1038/s41598-017-06761-4

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© The Author(s) 2017

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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Time-course DNA microarray analysis of 3T3-L1 cells treated with MDI plus DMSO or MDI plus echinomycin. (A) Clustering analysis performed among the MDI + DMSO treated groups using 2,204 selected probes that had a fold change ≥ 2.0 in expression compared with the control (0 hr) (See the details in Methods). Genes were clustered into 3 groups according to their timing of response to MDI treatment. (B) (a,b) Scatter plot of all the 14,005 probes (signal intensity ≥ 100 at any one point) at 2 hr and 6 hr. A log2-fold change from the control (time 0 hr) of the MDI + echinomycin group is plotted against a log2-fold change from the control (time 0 hr) of the MDI + DMSO group. (c) The scheme of probe selection for further clustering. Genes that had a fold change ≥ 2.0 (x ≥ 1) at both 2 hr and 6 hr relative to time 0 preadipocytes were further analysed using hierarchical clustering. (C) Representative quantitative RT-PCR analysis of Cebpb, Cebpd, Klf4, and Klf5 were performed to validate the microarray analysis. The data are the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, as determined by a two-way ANOVA with Bonferroni’s post-test. (D) A heatmap of the expression ratio (compared with 0 hr time point) is shown, and 111 probes are extracted as a cluster containing the genes induced by MDI and suppressed by echinomycin at both 2 hr and 6 hr.