HJURP-dependent CENP-A recruitment and incorporation into the LacO/TRE array. (A and B) Recruitment of endogenous CENP-A to the LacO/TRE array in the presence of LacI-HJURP or LacI-HJURP^Scm3. All LacI constructs have an N-terminal mCherry tag. Representative images of preextracted cells treated with 0 mM IPTG (A) or 10 mM IPTG (B) for 1 h before fixation. Endogenous CENP-A was detected using a monoclonal anti–CENP-A antibody. mCherry-LacI fusions of HJURP or HJURP^Scm3 and GFP-TetR markers are transiently transfected at equal ratios, and DNA is visualized using DAPI. Cells are fixed at 48 h after transfection. Arrows indicate the array. Insets show magnified views of boxed regions. (C) Quantification of CENP-A staining at the LacO/TRE array. Error bars represent the standard deviations between two experiments. At least 30 cells per condition were analyzed; n = 2. In the case of IPTG treatment, cells in which the residual mCherry signal was still visible at the array were excluded. (D) Quantification of the amount of CENP-A at the array in LacI-HJURP– and LacI-HJURP^Scm3–transfected cells with and without treatment with IPTG (>28 cells/condition). Middle lines in each box represent the mean integrated intensity for each condition, and whiskers represent the maximum and minimum intensities observed. A.U., arbitrary unit. Bars, 5 µm.