TPX2 phosphorylation rescues the short spindle defect caused by Aurora A depletion and specifically affects spindle length in metaphase. (A–C) HeLa cells were transfected with scrambled or Aurora A siRNA, or cotransfected with Aurora A siRNA and the indicated GFP-TPX2 constructs. Aurora A depletion causes short spindles, and TPX2-2D, but not -WT or -2A, could rescue such a defect. Bar, 10 µm. Three independent experiments were performed in C and n = 100, 94, 94, 60, and 88. Error bars indicate SEM. ***, P < 0.001. (D) His-TAT-RFP tagged TPX2-WT, -2A, and -2D purified from bacteria were visualized by Coomassie blue staining (left) and Western blotting with TPX2 antibody (right). The arrows indicate main bands of TPX2. (E–G) Cells treated with TAT-RFP-TPX2-2A show reduced length of metaphase spindles compared with normal structures in untreated, WT, or 2D-treated cells. Bar, 10 µm. n = 34, 76, 34, and 32. Error bars indicate SEM.