High levels of CENP-A in xenograft tumor tissues and in cultured DLD1 cells. (A) CENP-A–positive nuclei are higher in xenograft tissue derived from mouse fed with the DOX diet. Bar chart shows proportion of CENP-A–positive nuclei analyzed from IHC images as in Fig. 7 B. “N” denotes numbers of cells analyzed. P value was calulcated by using the χ² test. (B) Levels of YFP–CENP-A in tumor tissue of mouse on DOX diet. Western blots show levels of YFP–CENP-A in nuclear extracts collected from tumor tissues of MDX#07 or MDX#10 mice. (C) Dose-dependent OE of YFP–CENP-A in DLD1 cells. Western blots show protein levels of YFP–CENP-A in DLD1^CENP-A cells with or without 0.1 µg/ml or 1.0 µg/ml DOX treatment for 4 d. Blots were probed with anti-GFP antibody for expression of YFP–CENP-A, and anti-GAPDH antibody was used for loading control. (D) Higher mislocalization of YFP–CENP-A in DLD1 cells treated with 1.0 µg/ml DOX for 4 d. Images show YFP–CENP-A localization in DLD1^CENP-A cells treated as in A. Cells were immunostained with antibodies against CENP-A and stained with DAPI. (E) Reduced cell viability in cells with constitutive high OE of CENP-A. Brightfield microscopic images show viability status of DLD1^CENP-A cells treated as in C. (F) Total cell population and cell viability are reduced in DLD1^CENP-Acells treated with 1.0 µg/ml DOX. Bar charts show the proportion of total cells (left) and viable cells (right) in DLD1^CENP-A cells treated with indicated concentrations of DOX for 4 d. Error bars represent SEM from three independent experiments. P values calculated by using unpaired Student’s t test are indicated.