Figure 3.

Knockdown of Rack1 inhibits the proliferation, migration and invasiveness of multidrug-resistant breast cancer cells. (A) Western blotting analysis of P-gp, Rack1, c-Src and Anxa2 expression in MCF-7/ADR cells transfected with control or Rack1 specific stealth siRNAs (# represents siRNA sequence ); (B) Silencing of Rack1 expression inhibited cell proliferation compared with that of control cells. Wild type, control and Rack1 knockdown cells were seeded in 96-well plates at a density of 4 × 10³ cells per well and consecutively cultured for 24, 48, 72, 96 and 120 h. The cell proliferative activity was calculated using the CCK-8-based method by measuring the absorbance at 450 nm on a micro-ELISA reader. The assays were carried out using six replicates for each time point and repeated three times; (C) Knockdown of Rack1 induced a significant decrease in the colony formation ability of breast cancer cells. Control and Rack1 knockdown cells were seeded in 35-mm dishes at a density of 500 cells per dish and cultured for 10 days. The number of colonies was quantified under an inverted microscope; (D) Knockdown of Rack1 significantly inhibited cell migration as measured by the wound healing assay (100× magnification). Relative cell migration distance was quantified and plotted in the right panel. Data as the mean ± SD of triplicates, p < 0.0001; (E) Knockdown of Rack1 expression inhibited the invasion ability of breast cancer cells (200× magnification). Approximately 200 μL of cell suspension (approximately 8 × 10⁴ cells) in serum-free medium were added to the upper transwell inserts pre-coated with Matrigel; the lower insert was loaded with 10% FBS-containing cell culture medium. After 36 h of incubation at 37 °C, invaded cells were fixed, stained and counted under an inverted microscope. The average result of triplicate experiments is summarized in the right panel. Data as the mean ± SD, p < 0.0001 versus control cells. Statistical analysis was performed by one-way ANOVA (* p < 0.05).