A, moderate and stable overexpression of Pin1, and its W34A mutant and K63A mutant in HMLE cells using retrovirus-mediated gene transfer, assessed by immunoblot.
B and C, overexpression of Pin1, but not its W34A or K63A mutant increased mammosphere-forming activity in HMLE cells. Scale bars, 100 μm.
D and E, overexpression of Pin1, but not its mutants, in HMLE cells potently induced expansion of BCSCs, as assayed by FACS analysis of the BCSC-enriched CD24−CD44+ population.
F and G, Pin1 overexpression increases tumorigenicity of BCSCs. Transformed HMLE (HMLE-Ras) cells stably infected with control vector and Pin1 were injected into subcutaneous sites of nude mice in limiting dilutions. Two months later, mice were sacrificed and evaluated for tumor weight (F) and frequency (G). Pin1-overexpressing HMLE-Ras cells exhibited significantly much higher tumor incidence and grew much faster than control cells.
In all panels, error bars represent SD.