Constitutive high levels of CENP-A OE contribute to apoptosis in DLD1 cells. (A) Constitutive low levels of CENP-A OE do not induce apoptosis in DLD1^CENP-A cells. Flow cytometry analysis and quantification show the proportion of cells that are positive for Annexin and/or PI in DLD1^CENP-A or parental DLD1 cells with or without 0.1 µg/ml DOX treatment for 4 d. DOX was replenished every 2 d for constitutive overexpression of YFP–CENP-A. (B) Constitutive high levels of CENP-A OE induces apoptosis in DLD1^CENP- cells. Flow cytometry analysis and quantification show a high proportion of cells that are positive for Annexin and/or PI in DLD1^CENP-A cells with 1.0 µg/ml DOX for 4 d compared with parental DLD1 cells without DOX treatment. Live cells were stained with Annexin V or PI and analyzed in flow cytometry. Error bars represent SEM from three independent experiments. P values were calculated by using unpaired Student’s t test.