Figure 3

Effect of GSK3β and CRM1 inhibition on TSA induced cyclin D1 degradation. A, MCF-7 cells were transfected with or without GSK3β specific siRNA pools. 72 h after transfection, cells were treated with TSA (1 μM) alone or in the presence of MG132 (50 μM) for 6 h. Cell lysates were separated by 4–20 % SDS-PAGE and immunoblot analysis was done using antibodies against GSK3 α/β, cyclin D1 and actin. B, Effect of TSA on GSK3β levels and activity. MCF-7 cells were cultured in the absence or presence of TSA for 6 h. Immunoblot analysis was done with antibodies against GSK3, cyclin D1 and actin. C, partial inhibition of TSA induced cyclin D1 degradation by the GSK3 inhibitor SB216763. Cells were cultured for 24 h with the indicated concentrations (μM) of SB216763 and then treated for 6 h with TSA (1 μM). Immunoblot analysis was done with antibodies against β-Catenin, cyclin D1 and actin. D, Effect of SB216763 on GSK3β mRNA expression. MCF-7 cells were cultured for 24 h in the absence or presence of SB216763 (10 μM) and treated with TSA (1 μM) 12 h. Semi-quantitative RT-PCR analysis was done with primers for GSK3β, cyclin D1 and CGI-128 as a loading control. E, effect of the CRM1-dependent nuclear export inhibitor Leptomycin B (LMB) on TSA induced cyclin D1 degradation. MCF-7 cells were pretreated for 1 h with the indicated concentrations (ng/mL) of LMB and then treated with TSA (1 μM) for 6 h. Alternatively MCF-7 cells were cultured in the presence of the indicated concentrations (ng/mL) of LMB for 6 h. Immunoblot analysis was done with antibodies against cyclin D1 and actin.