CENP-C and CAL1 are required for centromeric localization of newly synthesized CID. (A) Centromeric localization of newly synthesized SNAP-CID is defective after CENP-C or CAL1 depletion. Localization of DNA (DAPI), total SNAP-CID (SNAP Ab), and newly synthesized SNAP-CID (TMR*) are shown from representative images of cells treated with DOTAP transfection reagent only (control, top), CENP-C dsRNA (CENP-C RNAi, middle), and CAL1 dsRNA (CAL1 RNAi, bottom), respectively. Total SNAP-CID (SNAP Ab) and newly synthesized SNAP-CID (TMR*) overlap in a typical punctate centromere pattern in control cells that is not visible in CENP-C– or CAL1-depleted cells. Each image in the right panels shows an individual cell from the merge panels (dashed circle) at increased magnification. (B) Quantification of frequencies of cells with different intensities of TMR* labeling at centromeres in control cells or after CENP-C (CENP-C RNAi) or CAL1 depletion (CAL1 RNAi). Centromeric TMR* signal intensities were categorized into strong (green), medium (yellow), weak (red), or no signal (blue). The total numbers of cells used in the analysis are indicated above each graph. Most control cells display strong to medium TMR* signals at centromeres, representing normal centromere localization of newly synthesized CID. In contrast, the majority of cells contained weak or no TMR* signal after CENP-C and CAL1 RNAi, suggesting defective localization of newly synthesized CID and a role for these proteins in CID assembly. Bars, 15 μm.