Figure 1

Effect of CGK733, caffeine and KU55933 on cyclin D1 stability. A. MCF-7 breast cancer cells were cultured with 10 μM CGK733 for the indicated times. Total lysates were resolved by SDS- PAGE and analyzed for cyclin D1 expression. Gel loading was monitored with an antibody raised against Hsp60. B. MCF-7 cells were cultured with the indicated concentrations of CGK733 for 6 h and analyzed as in A. C. T47D breast cancer cells were treated as in B. D. MCF-7 cells were incubated with 10 μM CGK733 alone or in the presence of 40 mM LiCl or 25 μM MG132 for 6 h and analyzed for cyclin D1 expression. Gel loading was monitored with an antibody directed against α- Tubulin. E. T47D cells were treated as in D. F. MCF-7 cells were grown on coverslips and treated with 10 μM CGK733 ± 25 μM MG132 for 6 h. Cyclin D1 expression was determined by indirect immunofluorescence microscopy as described in Materials and methods. Scale bar 10 μm. G. LnCap prostate cancer cells were treated and analyzed as in D. H. MCF-7 cells were cultured with 5 mM caffeine ± 25 μM MG132 for 6 h and analyzed as in D. I. MCF-7 cells were cultured in the presence of 5 mM caffeine, 10 μM CGK733 or 20 μM KU55933 for 24 h and analyzed for RB and cyclin D1 expression. J. MCF-7 cells were cultured with the indicated concentrations of CGK733 for 24 h and analyzed as in I.