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. Author manuscript; available in PMC: 2016 Dec 17.
Published in final edited form as: Mol Cell. 2015 Nov 19;60(6):886–898. doi: 10.1016/j.molcel.2015.10.027

Figure 2. Inducible knockouts reveal interdependencies between CCAN components.

Figure 2

A) Representative immunofluorescence images of control mitotic cells and CENP-N knockout mitotic cells stained for selected CCAN components. Images are deconvolved and scaled equivalently for each antibody. The centromeres of unprocessed images of this type were quantified to generate the data in panels B–F. See Fig. S2A for corresponding images from these cells showing DNA staining. B–F) Mean centromeric fluorescence intensity of a component of each CCAN subcomplex in mitotic cells following knockout of the indicated CCAN subunit for five days, normalized to cells in which spCas9 is not induced. n = 20 cells per condition per antibody per knockout, error bars represent s.e.m. For CENP-C, -N, -I, and –T knockouts, cells exhibiting mitotic errors were quantified. For the CENP-O knockout, which does not exhibit a mitotic phenotype, mitotic cells were selected at random. G) Summary of interdependencies of CCAN components determined by the inducible knockout strategy (Figures 2B–F). “+” indicates fluorescence intensity following knockout > 20 % of control; “−” indicates fluorescence intensity following knockout < 20 % of control. H) Summary of functional relationships between CCAN components determined using the inducible knockout strategy. Scale bar, 5 μm.