DOX-induced overexpression of CENP-A does not contribute to cell-cycle defects in DLD1^CENP-A cells. (A) CENP-A OE does not lead to defects in cell-cycle progression. Flow cytometry analysis of DLD1^CENP-A cells with or without DOX treatment shows similar cell-cycle profiles with 2N (G1/S stage) and 4N (G2/M stage) DNA content. (B) CENP-A OE does not contribute to reduced cell proliferation. Flow cytometry plot showing the distribution of EdU-stained DLD1^CENP-A cells with or without DOX treatment in different cell-cycle stages. Results were confirmed by using three biological repeats. Tables show the absolute percentage of cells in different cell-cycle stages. (C) DOX treatment does not increase chromosome segregation defects in parental DLD1 cells. Images show chromosome segregation status in parental DLD1 cells with or without DOX treatment. Cells were immunostained with antibodies against CENP-A and stained with DAPI. Cells were then analyzed for chromosome segregation outcome. Scale bar: 5 µm. (D) Quantification shows the proportion of cells with defective segregation in parental DLD1 cells treated as in F. Error bars represent SEM from three independent experiments. “N” denotes number of cells analyzed. P values calculated by using the unpaired Student’s t test are indicated.