Figure 5.

Skp2 is necessary, but not sufficient for TIS. Band intensities are normalized to α-tubulin and expressed in relative units. (A) Over-expression of Skp2 in PC3 cells. PC3 cells were incubated overnight after coelectroporation of GFP with either LacZ or Skp2 expression vectors. Cells were harvested 3 days after electroporation. (B) Overexpression of Skp2 disrupts the senescence response to AZQ. PC3 cells were electroporated, allowed to recover overnight, incubated in drug-free medium (-) or medium + 250 nM AZQ for 2 days, and analyzed for both protein expression and DNA content/cell cycle phase. The overexpression of Skp2 inhibits p27 induction and prevents the decrease in S-phase seen after AZQ treatment. Similar results were demonstrated in DU145 (not shown). (C) Persistent down-regulation of Skp2 expression inhibits proliferation in LNCaP but not DU145 and LNCaP + E6 cells. Skp2-specific shRNAs were expressed in LNCaP, DU145 (DU), and LNCaP + E6 cells (LN + E6). Cells were cultured for 7 days in drug-free medium (-) or with 2 µg/ml doxycycline (DOXY) (+), for 14 days with 2 µg/ml doxycycline (++), or for 7 days with 2 µg/ml doxycycline followed by 7 days in doxycycline-free medium (+-). Parallel samples were analyzed for protein expression and DNA content/cell cycle phase. Skp2 down-regulation inhibits proliferation in LNCaP cells but not DU145 and LNCaP + E6 cells. Student's t test, *P <.05, **P <.01.