Figure 1: MAN2C1 negatively regulates PTEN function.

(a) LNCaP cells were infected with EV (empty vector)/PTEN or MAN2C1/PTEN and selected with puromycin for PTEN-retrovirus infection. Surviving cells were stained with crystal violet (top left panels) and quantified (top right panel). Ectopic proteins were demonstrated by western blotting (bottom panel). (b) DU145 cells were infected with a retroviral-based PTEN siRNA or control siRNA (Ctrl siRNA1), followed by transfection with 100 nM MAN2C1 siRNA or its Ctrl siRNA2 for 3 days. The expression of AKT-P (Ser473 phosphorylated AKT), AKT (total AKT), MAN2C1, PTEN and actin were determined by western blot. The levels of individual proteins were quantified against actin and presented under individual panels as fold changes in comparison to Ctrl siRNA-treated cells. (c) Five days after transfection with MAN2C1 siRNA, cell numbers were counted and expressed as percentages of Ctrl siRNA-treated cells. Experiments were repeated three times. Means±standard derivations are shown. *Statistical significance (two-tailed Student's t-test) in the indicated comparisons (Ctrl siRNA versus MAN2C1 siRNA, P<0.001; MAN2C1 siRNA versus MAN2C1 siRNA/PTEN siRNA, P=0.001). (d) DU145 cells were infected with EV or MAN2C1 retrovirus and selected with hygromycin for 48 h to ensure 100% infection. Cells were then treated with DMSO (−) or wortmannin (Wort) for 1 h before western blotting. Experiments were repeated three times and a representative blot is shown. Levels of individual proteins were quantified against actin and presented under individual panels as fold changes in comparison to mock-treated EV cells. (e) LNCaP and U87 cells were infected with EV or MAN2C1 retrovirus, followed by selection with hygromycin for 3 days to achieve 100% transfection and then examined for AKT activation (AKT-P), AKT, ectopic MAN2C1 (using M2 anti-FLAG antibody) or actin by western blotting.