Figure 2. Human centromeres contain 400 molecules of CENP-A.
(A) Schematic outline of strategy allowing for the quantification of the centromeric fraction of CENP-A compared to the total cellular pool. Scale bars: 5 μm. (B) Quantification of the centromeric fraction of CENP-A in CAY/− cells. Each circle represents one centromere; circles on the same column are individual centromeres from the same cell. Dashed line indicates average of all centromeres. (C) Quantification of the centromeric fraction of CENP-A in indicated cell lines. Each square represents the average centromeric signal from one cell; squares on the same column are individual cells from the same experiment (Exp). Figure 2—figure supplement 2 shows quantification of individual centromeres in CAG/− and CAY/−+OE cells. (D) Representative quantitative immunoblot of purified recombinant CENP-A and endogenous CENP-A from whole cell extracts (WCE). (E) Quantification of D. Solid line represents the best fit linear regression. Dashed line represents the amount of CENP-A from 150,000 cells. (F) Quantification of the total cellular CENP-A copy number. Each diamond represents one replicate experiment; measurement from E is indicated as a gray diamond. (G) Calculation of average CENP-A copy number per centromere (CEN) in wild-type RPE cells. Throughout, the average ± SEM is indicated.
Figure 2—figure supplement 1. Representative fluorescence lifetime imaging (FLIM) micrograph of a CENP-A-YFP expressing cell (left) and quantification of indicated cellular regions (right).
Figure 2—figure supplement 2. Measurements of individual centromeres and CENP-A levels for different cell lines.
Figure 2—figure supplement 3. Transfer efficiency of recombinant and cellular CENP-A.
