Figure 5

Juglone, a Pin1 inhibitor, prevents exit from mitosis and dephosphorylation of CC-3 and MPM-2 mitotic antigens. Mitotic HeLa cells were withdrawn from nocodazole-containing medium and incubated for 5 hours in the absence (lane 2) or in the presence of juglone at 10 (lane 3) or 20 μg/ml (lane 4). The cells were then solubilized in sample buffer and processed for immunoblotting with CC-3, MPM-2 and POL3/3, a monoclonal antibody recognizing both hypo- and hyperphosphorylated forms of Rpb1. Lane 1 represents a sample of the cells removed before the 5-hour incubation and thus corresponds to t = 0. The juglone treatment prevented the dephosphorylation of the MPM-2 and CC-3 antigens after removal of nocodazole. The POL3/3 immunoblot revealed that Rpb1 became hyperphosphorylated after incubation with juglone (lanes 3 and 4) whereas the 5-hour chase in the absence of juglone (lane 2) allowed the cells to recover the usual 1:1 ratio of hypo- to hyperphosphorylated forms of Rpb1.