FIG. 7.
Pin1 inhibition is complemented by overexpression of a constitutively active cyclin D1. (A) Pin1 is essential to maintain cyclin D1 level and activity in Neu/Ras-transformed MCF-10 cells. MCF-10/Neu/Ras cells transfected with either GFP, GFP-Pin1, or GFP-dnPin1 were lysed and immunoblotted with anti-cyclin D1, -tubulin, and -GFP antibodies. (B) The same cell lysates as in panel A were immunoprecipitated with anti-cyclin D1 antibodies, followed by the in vitro kinase assay with GST-pRB as a substrate. Phosphorylation of the GST-pRB substrate is shown in the upper panel. The lower panel shows input GST-pRB stained with Coomassie blue. (C) MCF-10/Neu/Ras cells were transfected with either dnPin1 and pCDNA vector or dnPin1 and the cyclin D1T286A mutant (1:10 ratio) and selected with puromycin for 48 h. Cells were subjected to immunoblotting analysis with anti-HA and anti-GFP antibodies. (D) Cells were transfected as described for C and seeded on plastic plates for 3 weeks. Cells were fixed and stained with crystal violet. (E and F) Cells were transfected as described for panel C and cultured in 0.3% soft agar for 3 weeks. The number of colonies formed was scored. Representative phase pictures are shown in panel E. Colony numbers are the mean ± SD of three independent experiments (F).