Inhibition of p-Akt by TPX2 gene silencing in colon cancer cells. (A) The expression of p-Akt, total Akt, p-ERK-1/2, and ERK-1/2 on SW620 cells treated with shRNA-TPX2, shRNA (control), or Oligofectamine alone (Mock) was analyzed by western blotting. Blocking Akt activation decreases the colony formation induced by TPX2. SW480 cells were transfected with pcDNA3.1 or pcDNA3.1-TPX2 and then treated with LY294002 (25 μmol/L) for 2 h. (B) p-Akt and total Akt was analyzed by western blotting. (C) The colony formation ability of the cells was determined by a colony formation assay. The bars indicate mean colony numbers from a representative experiment done in triplicate; with SD. P < 0.05 compared to the no-treatment group. (D) Cell growth curve was measured by a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Each point indicates the mean of the spectrometric absorbance ± S.D. of three independent experiments (P < 0.01) (E) Immunofluorescence microscopic analyses of Akt activation. SW620 cells were infected with shRNA-TPX2 or control cells (control shRNA and mock). Then, the cells were immunostained for Akt (green) and their nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue).