Functional characterization of the PE enhancer. A, Luciferase reporter activity in JURKAT T-ALL cells of a pGL3 promoter empty construct (pGL3-Luc), a pGL3 promoter plus the human PE enhancer in the forward (hPE(+)-Luc) or reverse (hPE(−)-Luc) orientation, or a pGL3 promoter plus the mouse PE enhancer in the forward (mPE(+)-Luc) or reverse (mPE(−)-Luc) orientation. Data from three independent electroporation replicates are shown. B, Genotyping of DND41 single cell colonies harboring a heterozygous deletion for the PE enhancer. DND41 cells not electroporated (control) or electroporated with Cas9 but without gRNAs (no sgRNA) are shown as controls. C-E,
PTEN mRNA expression levels (C) and PTEN protein expression levels via intracellular FACS staining (D) or via western blot analysis, together with AKT activation (E) in DND41 control cells or two independent DND41 single-cell colonies with PE heterozygous deletion. F, Proliferation curve of DND41 control cells or two independent DND41 single-cell colonies with PE heterozygous deletion. **P < 0.01; and ***P < 0.005 values calculated using two-tailed Student’s t-test.