Fig. 1

MYCN overexpression antagonises RA-induced differentiation of neuroblastoma cells at the transcriptional level. a SY5Y-MYCN cells treated for the following time-periods: 1 μl/ml DMSO for 3 days, 1 μg/ml Dox (to induce MYCN expression) for 4 days, 1 μM RA for 3 days or combination treatment 1 μg/ml Dox for 4 days and 1 μg/ml RA for 3 days. Left: Imaging of the SY5Y-MYCN cells. All panels are imaged at 40× magnification. Right: The differentiation ratio of cells treated with individual agents or combination treatments was calculated by dividing the length of the longest axon of a cell by the cell’s width. The images from three biological replicates were pooled, and then measurements of individual cells were made using ImageJ v1.44p (http://imagej.nih.gov/ij). The range of measured cells (N) per treatment group is 150–259. The differentiation ratio of each cell was then calculated by dividing the length of its longest axon by the cell’s width. The p value for each treatment group compared with untreated control cells (t-test) is shown above each treatment box. b Number and overlap of differentially expressed (DE) genes in each treatment group, as detected by RNA-seq. The area-proportional Venn diagram was generated using BioVenn (http://www.cmbi.ru.nl/cdd/biovenn/) [123] and shows the overlap between the genes DE in each treatment group compared to the untreated control cells. c Proportions of up- and downregulated DE mRNAs for each treatment group when compared with untreated SY5Y-MYCN cells, as detected by RNA-seq. d All significant DE genes (RNA-seq), with expression level in the control state (no RA and un-induced MYCN) plotted against the fold change after treatment. Each significant DE gene is denoted by a purple dot