Evi1 down-regulates PTEN expression in BM cells. (A) Schematic representation of gene expression analysis. Evi1-GFP– or GFP-transduced BM cells (n = 4 for each) were sorted and subjected to gene expression analysis. Representative fluorescence-activated cell sorting data show the gene transfer efficiencies of 78% and 90% for Evi1-GFP and GFP-expressing retrovirus, respectively. (B) Real-time PCR for PTEN expression. Error bars indicate SD (n = 6; *P < .0001). (C) PTEN mRNA expression in lineage⁻, c-Kit⁺, Sca-1⁺ cells derived from Mx-Cre Evi1^flox/flox mice. Error bars indicate SD (n = 3; *P < .05). Evi1 was retrovirally added back. The Cre-mediated Evi1 deletion and the expression of wild-type Evi1 were evaluated by PCR with the use of genome DNA and cDNA, respectively, and representative figures are shown. Detailed experimental methods are shown in supplemental Data. (D) Schematic representation of mouse PTEN promoter region, possible Evi1 binding site predicted by rVISTA 2.0 (http://rvista.dcode.org/), constructs cloned into pGL4.10[Luc2], mutagenesis strategy, probe sets used for EMSAs, and primers for ChIP assays. (E) Relative luciferase activity of Evi1 on each PTEN promoter. Error bars indicate SD (n = 6; *P < .01). Jurkat cells were used. Error bars indicate SD. (F) Protein expression of Flag-tagged wild-type (WT) Evi1 and Evi1_R205N used for EMSAs. Western blot analysis was done with anti-Flag antibody. (G) Results of EMSAs are shown. Probe sets 3 or 3_mut are indicated in Figure 1D. Corresponding cold-specific competitors or nonspecific competitors were added as indicated. KO indicates knockout.