Optimization of DOX treatment and CENP-A expression. (A) Levels of endogenous CENP-A in different cell lines. Western blot shows levels of endogenous CENP-A in different cell lines as indicated. Blot was probed with anti–CENP-A antobody. GAPDH was used as a loading control. Three biological repeats were performed. (B) Expression of CENP-A in DOX-treated DLD1^CENP-A cells. Western blot shows levels of YFP–CENP-A in DLD1^CENP-A cells treated with 0.1 µg/ml DOX and after various time points post-DOX wash. Blot was probed with anti-GFP antibody. GAPDH was used as a loading control. (C) Quantification of endogenous and exogenous CENP-A levels. Bar chart shows the levels of endogenous CENP-A in different cell lines as in A and the levels of exogenously OE CENP-A in DLD1^CENP-A cells treated with DOX for 30 min followed by DOX removal for 20 h as in B. The levels of CENP-A normalized against the loading control, GAPDH, were compared as fold increase. Error bar represents SEM from two independent experiments. (D) Flowchart showing the experimental regimen for treatment with DOX used for all studies unless otherwise described. (E) CENP-A signal intensity is higher in DOX-treated DLD1^CENP-A cells. Violin plot showing the spectrum of CENP-A signal intensity across individual DLD1^CENP-A cells with or without DOX treatment. P values calculated by using the unpaired Student’s t test are indicated.