FIG 3.

The enzymatic activity of EST was required for the adipogenic effect of EST. (A) The activity of the EST AAK mutant was compared to that of the wild-type EST. 293T cells were transfected with a tk-ERE-Luc reporter gene and ERα, along with EST, EST AAK, or empty vector as indicated. The cells were either treated with 1 nM E₂ or vehicle in phenol red-free DMEM containing 10% DCC FBS. The luciferase activities are normalized against β-Gal activities from the cotransfected CMX-β-Gal vector. Results are shown as fold induction over vehicle, treated in triplicate. (B) Preadipocytes were infected with EST- or EST AAK-expressing lentivirus. The expression of EST and EST AAK was measured by Western blotting. (C to E) Preadipocytes were transduced with vector- or AAK-expressing lentivirus and then induced to differentiate for 14 days before being evaluated for oil red O staining (C), gene expression analysis by real-time PCR (D), and Western blot analysis to detect total AKT, phospho-AKT, total ERK1/2, and phospho-ERK1/2 (E). *, P < 0.05; ns, statistically not significant.