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. 2017 Mar 15;130(6):1078–1093. doi: 10.1242/jcs.196790

Fig. 5.

Fig. 5.

Impact of Twist1 phosphorylation on its oncogenic functions. (A) Domain stricture of Twist1. (B–E) The 3A-Twist1 mutant favors heterodimerization with E12 and Hand2. E12 and Hand2 retrovirus were infected in BxPC3 cells expressing either HA-tagged wild-type (WT) Twist1 or 3A-Twist1. After 36 h, Twist1 was immunoprecipitated (IP) and E12 and Hand2 binding analyzed by immunoblotting (IB). Similarly, Myc–E12 and Flag–Hand2 were immunoprecipitated, and Twist1 binding analyzed by using anti-HA antibody. The indicated ratios for a representative experiment are given below the blots. (F) Twist1 promotes cell proliferation in BxPC3 cells. (G) AURKA depletion decreases cell proliferation in BxPC3 and Twist1-BxPC3 cells, but not in phospho-dead 3A-Twist1-BxPC3 cells. *P<0.05, **P<0.01 compared to scrambled shRNA controls (two-sample t-test). (H) AURKA overexpression increases cell proliferation in vector-expressing BxPC3 and Twist1-BxPC3 cells, but not in 3A-Twist1-BxPC3 cells. An MTT assay was performed after 48 h. *P<0.05 compared to vector infected controls (two-independent sample t-test). (I) Twist1 promotes colony formation in a soft agar assay. *P<0.05 compared to vector-expressing control (two-sample t-test). (J) Twist1 promotes cell motility. **P<0.01 compared to vector-expressing control (two-sample t-test). (K) AURKA depletion inhibits cell motility in Twist1-BxPC3 cells, but not in phospho-dead 3A-Twist1-BxPC3 cells. Twist1-BxPC3 and 3A-Twist1-BxPC3 cells were infected with either scrambled shRNA or AURKA shRNA lentivirus. The cell migration test was performed after 30 h. **P<0.01 compared to scrambled shRNA control (two-sample t-test). (L) AURKA overexpression increases cell motility in Twist1-BxPC3 cells, but not in 3A-Twist1-BxPC3 cells. Twist1-BxPC3 and 3A-Twist1-BxPC3 cells were infected with either vector or AURKA retrovirus. Cell migration was performed after 30 h.