(A) Fluorescence microscopy analysis comparing kinetochore localization of a wild-type CENP-N-mCherry fluorescent reporter in human HeLa FlpIn TRex cells depleted of CENP-C and, were indicated, further expressing wild-type GFP-CENP-C or the 5A mutant. (B) Quantification of CENP-C (left) and mCherry-CENP-N (right) levels at kinetochores in mitotic cells following the rescue of CENP-C depletion by either GFP-CENP-CWT or the GFP-CENP-C5A mutant. Graphs show kinetochore fluorescence intensity of the indicated protein (antibodies against CENP-C or mCherry) normalized to CENP-C or mCherry-CENP-N kinetochore levels in the absence of RNAi treatment, respectively. Each graph is representative of two independent experiments. (C) Surface representation of a composite model built by combining the coordinates of the CENP-C motif (residues 712 to 733 from PDB ID 4 × 23, describing its interaction with nucleosome) with those of the CENP-N:CENP-ANCP complex. (D) Schematic of crucial kinetochore interactions, already shown in Figure 1A, but with question marks removed at interactions investigated in the present work. (E) The grey box, an enlargement of the box in D, summarizes the details of the interactions reported in this work, as well as previous information on the interaction of CENP-C with the CENP-ANCP.