Figure 3

Salinomycin downregulates cyclin D1 and Skp2 and accumulates p27Kip1. (a) Cells were treated without or with 4 μM salinomycin for 24 h, and equal amounts of cell lysates were subjected to immunoblot analysis using the indicated antibodies. exp., exposure. (b) DXR cells were treated without or with 4 μM salinomycin for 24 h, and the levels of cyclin D1 and Skp2 mRNA were determined by qPCR. ***P<0.0005. (c) DXR cells were treated without or with 4 μM salinomycin for 24 h, and 10 μM CHX was added for the last 1, 2, and 4 h before harvest, followed by immunoblot analysis using cyclin D1 and Skp2 antibodies. exp., exposure. (d) The graph shows the quantitation of immunoblot analysis data shown in panel c. Data are presented as averages of triplicate experiments, with error bars representing standard deviations. **P<0.005. (e) DXR cells were treated without or with 4 μM salinomycin in the presence or absence of 10 μM MG132 for 18 h, and equal amounts of cell lysates were subjected to immunoblot analysis using cyclin D1 and Skp2 antibodies. GAPDH was used as a loading control. (f) DXR cells were treated without or with 4 μM salinomycin for 18 h, and cell lysates were subjected to immunoprecipitation using anti-Skp2 or normal rabbit IgG antibodies, followed by immunoblot analysis for ubiquitin and Skp2. Similar results were observed in independent experiments