CENP-C targeting can initiate ectopic kinetochore assembly. (A) Metaphase chromosomes from lacO cells expressing GFP–LacI–CENP-C or CAL1–GFP–LacI (both in green) showing that CENP-A (red) is recruited at the lacO array (arrow) by GFP–LacI–CENP-C. (B) Quantification of the recruitment efficiency of CENP-A and Ndc80 by LacI–GFP–CENP-C; one (out of three total) representative experiment is shown (n_CENP-A = 77, n_Ndc80 = 86 cells). (C) Scatter dot plot of the CENP-A IF signal ratio between the lacO array and the endogenous centromere in the GFP–LacI–CENP-C and CAL1–GFP–LacI targeting experiments shown in A. Each dot represents the signal ratio for one lacO-containing chromosome (n = 20 cells per condition). Blue line, average ratio; magenta bars, standard deviation. Data are from a single representative experiment out of two repeats.