DLD1^CENP-A xenograft tumors with high expression of CENP-A exhibit abnormal mitoses and increased incidence of micronuclei. (A) Tumor volume in mice xenografted with DLD1^CENP-A cells and fed with CON or DOX diet. Ten mice that were s.c. injected with DLD1^CENP-A cells were fed with CON diet and tumor volumes were measured every 3–5 d. After 16 d of s.c. injection, five mice were continued on CON diet and the other five were fed with DOX diet (200 mg/kg ad lib) to overexpress YFP–CENP-A. Graph shows tumor volume in mm³ in mice fed with DOX diet (n = 5) or those that continued on CON diet (n = 5). (B) Higher expression of YFP–CENP-A in xenograft-induced tumor tissue of mouse fed on DOX diet. Images of IHC show YFP–CENP-A expression in tumor tissues of mice with DOX (MDX#07) or CON (MDX#10) diet. (C) Higher degree of abnormal mitoses in xenograft-induced tumor tissue of mouse on DOX diet. H&E-stained histological images show anaphases in tumors from mice with DOX (MDX#07) or CON (MDX#10) diet. Arrows in anaphase cells show abnormal anaphases as lagging chromosomes and DNA bridges. (D) Quantification of experiment (C) shows proportion of abnormal anaphases in H&E-stained tumor tissues from mice with DOX (MDX#07) or CON (MDX#10) diet. P value was calculated by using the χ² test. (E) Increased incidence of micronuclei in cells derived from xenograft-induced tumor from mouse fed with DOX diet. Images show micronuclei in cells derived from tumors of mice with DOX (MDX#07) or CON (MDX#10) diet. Cells were immunostained with antibodies against CENP-A, stained with DAPI, and analyzed for the presence of micronuclei in YFP-positive and -negative cells. Yellow arrows show micronuclei in cells derived from tumor of mouse fed on DOX diet. Scale bar: 5 µm. (F) Quantification of experiment (E) shows the proportion of cells with micronuclei in cells derived as in C. “N” in D and F denotes the number of cells analyzed. P value was calculated by using the χ² test.