Figure 2. Rab25 confers growth advantage to luminal B breast cancer cells.

(A) Equal number of cells stably expressing CONTROL vectors and pcDNARab25HA or shRab25 were seeded at 50% confluence and cultured under low serum conditions for 72 hours. At each time point the cells were harvested to measure viability using MTT based assay. MCF7 pcDNA control (solid black line) and MCF7 non-silencing control (dashed black line) show baseline viability of MCF7 cells. Rab25 overexpressing cells is the solid gray line while shRab25 expressing cells shown by dashed grays line. Data represents three independent experiments. (B) Less than 50 luminal B cells of either MCF7 or T47D lines with stable overexpression of Rab25 were seeded in 6 well plates under low serum condition for measuring anchorage dependent growth. Plates were harvested after 10 days and number of colonies quantified. Each condition done in triplicate wells. Data represents over 4 independent experiments. (C) T47D cells transfected with control or siRab25 before plating in triplicate wells for anchorage dependent colony formation assay. Colonies harvested after 10 days and quantified. Data represents 3 independent experiments. (D) MDA231 cells with stable expression of Rab25 plated at less than 50 cells per well in 6 well plate and allowed to colonize for 10days. Data represents at least 3 independent experiments. (E) MCF10A cells with stable expression of Rab25 plated at less than 50 cells per well in 6 well plates and allowed to colonize for 12 days under low serum condition. Data represents at least 3 independent experiments. (F) Immunoblot showing MCF7 cells with endogenous Rab25 or stably expressing shRab25 that were treated with cyclohexamide and serum starved ON, followed by stimulation with 25nMol EGF for 6 hours. Top panel shows pERK signal over time in MCF7 cells expressing control shRNA and lower is data from MCF7shRab25. The densitometry representing pERK signal at each time point each time-point normalized to its loading control is shown.