Rescue of centromere assembly by APC inhibition. (A) CYCA and RCA1 disruption of CID localization is rescued by FZR depletion. Localization of DNA (top), CID (middle), and CENP-C (bottom) in Kc167 cells treated with CYCA, RCA1, CYCA + FZR, or RCA1 + FZR dsRNAs. Control cells were not treated with dsRNA. (B) Quantification of CID and CENP-C fluorescence intensity levels at centromeres after RNAi treatment. Quantification of FZY-depleted cells is also included, showing specificity of the rescue by FZR depletion (n = 2; error bars represent SEM). (C) Protein levels of CID (31 kD), CENP-C (160 kD), CAL1 (120 kD), and CYCA (60 kD) after RNAi depletion in Kc167 cells. Nuclear extracts from Kc167 cells treated with no dsRNA (control) or dsRNA specific for CID, CENP-C, CAL1, CYCA, RCA1, FZR, CYCA + FZR, or RCA1 + FZR were Western blotted with antibodies against CLD genes or tubulin (50 kD) as a control. (D) Model for the role of CLD genes in cell cycle regulation of centromere assembly. The green background indicates proteins that localize to the centromere, orange indicates that the proteins are not competent for centromere assembly, and double arrowheads indicate that the protein or complex is required for the centromere localization of CYCA and CID-, CAL1-, and CENP-C–interdependent assembly. The APC substrate could be CYCA (red arrow) or CID, CAL1, or an unidentified substrate, X (blue arrow). Bar, 15 μm.