The effect of AKT on E2-induced alternative splicing of CD44 minigene. A) CD44 minigene splicing assay in 293 cells. 293 cells were transfected with ERE-CD44 minigene (2 μg), indicated ERα constructs (2 μg) and either vector control pCDNA3 or pCDNA3-CA-AKT expression vector (0.5 μg). A day after transfection, cells were treated with ethanol or E2 for 24 hours. qRT-PCR was performed using primers that amplify exon-included or exon-skipped products. The ratio between exon-included and exon-skipped products in untreated ERα and CD44-ERE transfected cells was normalized to one and relative change in the ratio is presented. Ratio of >1 indicates elevated exon-inclusion whereas a ratio of <1 indicates enhanced exon-skipping. B) The effect of E2 on CD44 minigene splicing in MCF-7p and MCF-7AKT cells. Cells were transfected with ERE-CD44 minigene or HSV-CD44 minigene and assays were performed as in A. Statistically significant differences are indicated.