Regulation of AR protein level and cell growth via the MID1-α4/PP2A transcriptional regulator complex. MID1, α4 and PP2A form the core of a ribonuclear protein complex that enhances translation of associated mRNAs such as AR mRNA. Physical interaction of the complex components was confirmed in LNCaP cells overexpressing a flag-tagged MID1. MID1 and PP2A were co-precipitated in an α4 pull-down (α4); normal rabbit IgG was used as negative control (IgG). A representative western blot of two independent experiments is shown in (A). Disruption of the complex by MID1 or α4 protein knockdown mimics the effect of metformin. MID1 knockdown in LNCaP and LNCaP-abl cells or α4 in LNCaP cells resulted in a reduction of AR protein levels (B) and the inhibition of cell growth (C). In the AR-negative PC3 cells, MID1 overexpression enhanced, whereas MID1 knockdown inhibited cell growth (D). Successful knockdown or overexpression, respectively, was verified by western blot; inserts show representative fluoroscans (B, D). Luciferase (Luci) siRNA was used as negative control for siRNA transfections, empty vector for overexpression control. Cell numbers were determined after 10 days. Statistical significant differences are indicated as *, p < 0.05; **, p = <0.01 and ***, p < 0.001.