Fig. 6
Combined TBX2-MYCN signaling targets the FOXM1/E2F network. a Heatmap showing GSEA enrichment scores (with FDR < 0.1) for an in house compiled gene set collection containing all MYC target genesets from the “Hallmark V5.1” catalog (MsigDB) as well as publically available MYC(N) activity or target signatures upon knockdown of TBX2 in IMR-5/75, CLB-GA, knockdown of MYCN in IMR-5/75 or the combination of TBX2 and MYCN knockdown in IMR-5/75. Size of the circles indicate FDR value and the color indicates the NES score. Positive values (red) point at enrichment among the upregulated genes while negative values (blue) indicate enrichment among the downregulated genes. b Synergistic reduction of cell proliferation upon combined knockdown of MYCN and TBX2 in the IMR-5/75 cell line measured with time-lapse microscopy (every 2–3 h) using the IncuCyte device (one out of three biological replicates is shown). Error bars represent the s.d. of five technical replicates. Bliss independence score was used to calculate potential synergism at 120 h for both the shTBX2 #2 and #4 compared with the double MYCN and TBX2 knockdown. Excess over bliss score for shTBX2 #2 is 0.092, excess over bliss score for shTBX2 #4 is 0.342. Excess over Bliss scores > 0 indicates synergy, < 0 indicates antagonism. c Combined TBX2 and MYCN knockdown in the IMR-5/75 cell line results in an enforced G1-cell phase arrest with almost no cells in S-phase or G2/M phase left (one replicate out of 3 shown). d Cumulative distribution plot of the absolute log fold changes of shTBX2, shMYCN or shTBX2:shMYCN up- and downregulated genes. Dashed line represents the sum of the log fold changes of the shTBX2 and shMYCN condition for the respective genes in the geneset. The difference between the blue line and the dashed line indicates synergism effect on logFC for the genes in the genesets implicated in cell cycle and MYCN regulation (two-sided paired t-test: p < 2.2e−16)
