AURKA-mediated phosphorylation of Twist1 is crucial for EMT, the CSC phenotype and drug resistance. (A) Expression of wild-type (WT) Twist1 increases CD44, Slug and Snail promoter activities and decreases E-cadherin promoter activity, whereas expression of 3A-Twist1 decreases CD44, Slug and Snail promoter activities and increases E-cadherin promoter activity. Results are for three independent experiments performed in triplicate. *P<0.05, **P<0.01 compared to control BxPC3 cells (one-way ANOVA). (B) Twist1 expression increases the levels of EMT and CSC markers while decreasing E-cadherin levels. 3A-Twist1 expression decreases the levels of EMT and CSC markers while increases E-cadherin. (C) AURKA inhibition decreases the levels of EMT and CSC markers. (D) AURKA overexpression increases the expression of EMT and CSC markers, while the expression of 3A Twist1 in the AURKA-expressing cells rescues the phenotype. The indicated ratios for a representative experiment are given next to the blots in B–D. (E) Twist1 overexpression increases the sphere-forming ability in BxPC3 cells. (F) Twist1 overexpression increases drug resistance in BxPC3 cells. BxPC3 cells and BxPC3 cells expressing wild-type Twist1 and 3A-Twist1, were plated in 96-well plates overnight. Then gemcitabine (1 µM) was added and cells were cultured for another 24, 48 or 72 h. (G) Twist1 shRNA-mediated depletion sensitizes BxPC3 cells to AURKA inhibition with MLN8237 (1 µM, treated for 48 h). *P<0.05, **P<0.01 compared to scrambled shRNA control (two-sample t-test).