Table 1.
Scheme of ChIP-chip hybridization per sample.
Figure 1.
Promoter ChIP enrichment of DAAM1 and NUMB verified by quantitative real-time PCR analysis.
A and B). Quantitative PCRs with primers designed to include the conserved ETS binding sequence in the proximal promoter of DAAM1 and NUMB were applied. The DAAM1 and NUMB promoters were enriched 1.5-fold over IgG control in six ChIPs and in five ChIPs, respectively (one of two representative experiments is shown).
Figure 2.
The significant genes derived from 7 ChIP-chip microarray data are displayed with the use of MultiExperiment Viewer graphical interface.
The fold enrichment for 48 genes shared by at least 3 of ChIP-chips was uploaded to MultiExperiment Viewer to obtain a heat map representing the gene overlap of all ChIP-chips. Significantly enriched genes are >1.25 fold change with respect to the IgG control. The overlap of 48 unique genes shows that AML D, T-ALL and nBM share a greater number of enriched target genes. AML A-E are ordered with respect to ERG mRNA expression and genes are ranked according AML D P-Values.
Table 2.
ChIP-chip enriched genes.
Table 3.
Motif analysis of enriched gene lists.
Figure 3.
Biological pathways enriched in primary leukemia ChIP-chip analyses.
Displayed are the biological pathways significantly enriched in at least three of seven primary leukemia samples by ChIP-chip. The bars represent the negative logarithm function of Fisher’s P-value (P<0.05).
Figure 4.
ERG induced dephosphorylation of AKT(Ser473).
A histogram overlay of ERG overexpressing cells in K562 and Jurkat cells (red tinted peaks) stained for intracellular pan AKT (left side histograms) and phosphorylated AKT(Ser473, right side histograms). Non ERG expressing cells are represented by an untinted black lined peak. The histograms represent two relative AKT levels in duplicate experiments of two K562 Tet-on ERG clones. Jurkat transient transfections of pDom-empty and pDom-ERG were carried out in duplicate.
Figure 5.
ERG overexpression induced resistance to multi-kinase inhibitors. A)
K562 cells transduced with the inducible ERG expression vector (induced indicated as +DOX and uninduced indicated as –DOX) were treated with 10 µM Sorafenib in 5 parallel wells. Following 48 hours, cell proliferation was measured with WST-1 at 450 nm absorbance. Jurkat cells were transiently transfected with ERG expression vector, pDom-ERG, and the control vector pDom-empty. Twenty-four hours after seeding transiently transfected cells in 5 parallel wells, Sorafenib was added to a final concentration of 10 µM. Cell proliferation was measured 48 hours after drug addition. Cell proliferation was measured with WST-1 reagent at the 450 nm absorbance. Cell proliferation was measured with WST-1 in Jurkat cells transiently transfected with ERG expression vector pDom-ERG and the control vector pDom-empty. Twenty-four hours following transient transfection of pDom-ERG and pDom-empty, TKI258 was added to a final concentration of 1 µM. Cell proliferation was analyzed as described above. Bar graphs display the average of 5 experiments. Statistical significance was analyzed using Wilcoxon rang sum test. Asterisk indicates statistically significant results. *: P<0.05. B and C) ERG (+Dox and −Dox) K562 cells were treated with 10 µM Sorafenib or 10 µM TKI258 for 72 hours in order to determine the effects of drug induced apoptosis by Annexin V-FITC detection. This was conducted in two independent ERG inducible K562 Tet-on clones and one of two independent experiments is represented by dot plots.