FIGURE 6.

Mitotic phosphorylation of Ajuba is required for cell proliferation and anchorage-independent growth. A, HPNE cells stably expressing vector, Ajuba, or Ajuba-2A were established, and expression of Ajuba and Ajunba-2A were confirmed by Western blotting. 2A, S119A/S175A. B, cell proliferation assays with transduced HPNE cells established in A. Data were expressed as the mean ± S.D. of three independent experiments. ***, p < 0.001; *, p < 0.05 (Ajuba-WT versus Ajuba-2A) (t test). C, establishment of Tet-On-inducible Ajuba-knockdown cell lines expressing shRNA-resistant Ajuba or Ajuba-2A in RCA colon cancer cells (see ”Experimental Procedures“). Cells were kept on Tet-approved FBS and doxycycline was added (1 μg/ml) to the cells 2 days prior to the experiments. D, cell proliferation assays in RCA cells established in C in the presence of doxycycline (DOX). Data were expressed as the mean ± S.D. of three independent experiments. Red asterisks mark the comparisons between shControl and shAjuba. Green asterisks indicate the comparisons between Tet-Ajuba-WT and Tet-Ajuba-2A. ***, p < 0.001; **, p < 0.01; *, p < 0.05 (t test). E and F, colony assays in soft agar to assess anchorage-independent growth of RCA cells established in C in the presence of doxycycline. Data were expressed as the mean ± S.D. of three repeats (E) and representative images were shown (F). ***, p < 0.001; **, p < 0.01 (t test).