FIGURE 5.

Effect of knockdown of UbcH5c on cyclin D1 levels in MDA-MB-468 and MCF7 cells. A, immunoblot analysis showing the effect of knockdown of UbcH5c (H5cKD) on cyclin D1 level in MDA-MB-468 cells. Control cells were transfected with control siRNA. B, immunoblot analysis showing the effect of knockdown of UbcH5c on cyclin D1 level in MCF7 cells. β-Actin was used as loading control. Control cells were transfected with control siRNA. C, evaluation of cyclin D1 and UbcH5c protein levels in MDA-MB-468 cells with or without knockdown of UbcH5c. Six independent UbcH5c-knocked down cell populations and corresponding control siRNA-treated cells were used. D, evaluation of cyclin D1 and UbcH5c protein levels in MCF7 cells with or without knockdown of UbcH5c. Six independent UbcH5c-knocked down cell populations and corresponding control siRNA-treated cells were used. For the experiments in C and D, 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). E, proliferation assays with the control and UbcH5c knockdown MCF7 cells in the absence or presence of 4HT (10 μm) and the effects of simultaneous knockdown of cyclin D1 (CD1+H5cKD) in these processes. 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). F, Matrigel invasion assay with UbcH5c knocked down MCF7 and MDA-MB-468 cells. Results are mean ± S.E. (n = 6). Data with siRNA stealth-1106 (supplemental Table 2S) are shown. Other siRNA, stealth-1214, also yielded similar results (data not shown).