FIGURE 3.

Effect of SLUG expression on UbcH5c and cyclin D1 levels in MDA-MB-468 and MCF7 cells. A, a typical immunoblot showing UbcH5c and SLUG protein levels in different human breast cancer cells. MB468, MDA-MB-468 cells; MB231, MDA-MB-231 cells. B, real-time RT-PCR analysis of the levels of SLUG, UbcH5c, and cyclin D1 mRNAs in SLUG-overexpressing MCF7 and MDA-MB-468 cells. Results are mean ± S.E. (n = 6). The differences between the experimental and control sets were statistically significant (p < 0.001). C, immunoblot analysis for SLUG and UbcH5c proteins in the control and SLUG-overexpressing (SLUG) MCF7 and MDA-MB-468 cells. D, densitometric scan for SLUG and UbcH5c levels in six independent SLUG-transfected populations and corresponding vector-transfected control cells. Results are mean ± S.E. (n = 6). The -fold changes were statistically significant (p < 0.001). E, immunoblot analysis data showing the effects of SLUG overexpression on the levels of cyclin D1, phosphocyclin D1 (at Thr²⁸⁶), GSK3β, phospho-GSK3β, AKT, and phospho-AKT in MDA-MB-468 cells. Control cells (V) were transfected with empty vector DNA instead of SLUG construct plasmid DNA. Recombinant SLUG was FLAG-tagged at the C-terminal end and thus was detected with anti-FLAG antibody. β-Actin was used as a loading control. F, effect of cyclin D1 knockdown (CD1KD) on the SLUG-induced increase in cell proliferation and tamoxifen (4HT; 10 μm) resistance in MCF7 cells. Control cells were transfected with empty vector DNA instead of SLUG construct plasmid DNA. Results are mean ± S.E. (n = 6). Data with cyclin D1 siRNA stealth-311 (supplemental Table 2S) are shown. Other siRNA, stealth-568, also yielded similar results (data not shown). The ability of stealth-311 to knock down cyclin D1 in MDA-MB-231 cells is shown in supplemental Fig. 3S.