Figure 2

Effect of MIB on p27, SKP2, and other cell cycle regulators (A) LNCaP cells were incubated with 10 ng/ml MIB for the indicated periods. Cell lysate was harvested and expression of the indicated proteins was assessed by immunoblotting with respective antibodies. Tubulin is shown as a loading control. SKP2 and p27 protein levels were quantitated using a demonstration copy of the Totallab software from Nonlinear Dynamics. (B) LNCaP cells were incubated with the indicated concentrations of MIB for 72 h, followed by preparation of total cell lysate and immunoblotting with antibodies against p27, SKP2, and tubulin. The effects of androgen on p27 and SKP2 were maximal at 1 ng/ml MIB. (C) LNCaP cells were maintained in the presence of 1 ng/ml MIB for 96 h after which the medium was replaced and 750 ng/ml of the antiandrogen cyproterone acetate (CA) was added. Cell lysates were prepared after the indicated times, and p27 and SKP2 levels were assessed by immunoblotting. (D) LNCaP cells were incubated with 1 ng/ml MIB and/or 750 ng/ml CA for 72 h and cell lysates were prepared. Expression of the indicated proteins was assessed by immunoblotting. Tubulin levels are shown as loading controls. (E) LNCaP cells were incubated with 1 ng/ml MIB and/or 750 ng/ml CA for 72 h and cells were fixed for flow cytometry. Cell cycle profiles and the fraction of cells in each cell cycle phase are shown. (F) LNCaP cells were maintained in the absence or presence of 10 ng/ml MIB for 72 h. Cells were fixed and double-stained with antibodies against p27 (red) and SKP2 (green). Cell nuclei were counterstained with DAPI.