Quantification of CENP-A Localization

For quantification analysis of CENP-A (Fig. 2), z-stack images were acquired with a spacing of 0.22 µm to cover an entire nuclear signal on an Axio Observer.Z1 microscope (Zeiss) equipped with a CSU-X1 confocal scanner unit (Yokogawa), iXon3 DU897E-CS0 camera (Andor) and Plan-Apochromat 100×/1.46 oil lens (Zeiss) using Andor iQ2 software (Andor). Quantitative analysis of acquired images was performed as previously described (Shono et al., 2015 (link)). Other cell images were acquired on an Axio Observer.Z1 (Zeiss) equipped with a LSM700 scanning module and an Objective Plan-Apochromat 63×/1.46 oil lens (Zeiss) using ZEN 2009 software (Zeiss). For quantification analysis in Fig. 4, z-stack images were acquired with a spacing of 0.36 µm, and with total depth of 5.4 µm. Signal intensity was acquired for each pixel in a 22×22 pixel region around the spot from a z-stack maximum intensity projection cell image using Fiji software (Schindelin et al., 2012 (link)). The pixel intensities located in a symmetric position with respect to the x-axis and y-axis were summed up. The integrated pixel intensities at the same distance from the spot were averaged.