FIGURE 4.

Effect of knockdown of SLUG on cyclin D1 levels in MDA-MB-231 and BT549 cells. A, quantitative RT-PCR analysis for SLUG, UbcH5c, and cyclin D1 mRNA levels in MDA-MB-231 cells treated with different siRNAs (supplemental Table 2S). B, immunoblot analysis of UbcH5c and cyclin D1 levels in MDA-MB-231 and BT549 cells with (SLUGKD) or without (Control) knocking down SLUG (siRNA#1, stealth-21). Control cells were transfected with control siRNA. C, evaluation of SLUG, cyclin D1, and UbcH5c protein levels in MDA-MB-231 and BT549 cells with or without knockdown of SLUG. Six independent SLUG-knocked down cell populations and corresponding control siRNA-treated cells were used. Data with siRNA#1 as the reagent for the knockdown are shown. Similar results were obtained with stealth-223 (siRNA#2; data not shown). Bands were developed using IR dye-conjugated secondary antibody (LI-COR Biosciences) and visualized using the LI-COR Odyssey infrared imaging system. Quantitation and analysis of bands were performed using Odyssey software. β-Actin was used as normalization control. Results are mean ± S.E. (error bars) (n = 6). -Fold changes observed were statistically significant (p < 0.001). D, effect of knockdown of SLUG in MDA-MB-231 cells on their rate of proliferation and the role of non-degradable cyclin D1 mutant (T286A) in this process. Cells were transiently transfected with wild type or HA-tagged T286A mutant of cyclin D1 along with the siRNA (control or anti-SLUG). Results are mean ± S.E. (n = 6). The effect of SLUG knockdown with wild-type cyclin D1 was statistically significant (p < 0.0001). E, effect of the proteasome inhibitor MG132 on the decrease in cyclin D1 in SLUG-knocked down MDA-MB-231 cells. Control cells were transfected with empty vector DNA. β-Actin was used as loading control. Veh, vehicle (DMSO) for MG132 solution. F, densitometric analysis of immunoblot data as in E from three independent experiments. The upper panel (i) shows the effect on SLUG level, and the lower panel (ii) shows the effect on cyclin D1 levels. Experimental data are normalized assuming respective control as 100. Results are mean ± S.E. (n = 3); *, statistical significance in comparison with respective control (p < 0.001).