Figure 4.

Pin1 inhibits Rb function in cell cycle progression and senescence. (a) U2-OS (Rb-positive) or Saos-2 (Rb-null) cells were co-transfected with WT or mutant Pin1, or a vector control, along with an E2F1-responsive DHFR-Luciferase (DHFR-Luc) reporter and a β-Galactosidase expression plasmid. Cells were lysed 24 h after transfection and subjected to luciferase and β-Galactosidase activity assays. DHFR-Luc activity was normalized to β-Galactosidase activity as described in the Materials and Methods and presented as fold activation. Results presented as means and S.E. of three independent experiments performed in triplicate. **P<0.01. (b) H1299 cells were stably infected with shRNA against Rb, Pin1 or a control, and selected by puromycin resistance. Cell lysates were subjected to western blotting, as indicated. (c) Stable H1299 cells were subjected to flow cytometry for analysis of cell cycle stage. (d and e) Saos-2 cells were co-transfected with Rb and/or WT or mutant Pin1, or with a vector control, and selected by puromycin resistance, as described in the Materials and Methods. Stable cells were grown in normal growth media supplemented with 0.5 μg/ml puromycin for 10 days. Cells were then visualized under a light microscope for the analysis of large/flat phenotype (d) or were subjected to β-Galactosidase staining (e). Cells were scored and presented as the percentage of stained cells over total cells. Results are presented as representative images and mean and S.E. from three independent experiments. At least 300 cells were counted for each condition