Effect of single-stranded RNA-specific RNaseA treatment on the localization of centromere proteins on HeLa metaphase chromosomes. In total, 25 cells with (+) or without (−) RNaseA treatments, were scored using the Ip Lab program. For each cell, immunofluorescence intensities for five randomly chosen chromosomes were quantified. Each data set was normalized against the CREST signal (Aii–Hii; red) and expressed as “mean ratio.” The RNaseA treatment resulted in a significant reduction (P-value < 0.001) in the immunofluorescence signals for CENPC1 (Ciii,Diii; green), INCENP (Eiii,Fiii; green), and survivin (SURV) (Giii,Hiii; green), but not for CENPA (Aiii,Biii; green). As with the RNase treatment shown in Fig. 4, signal reduction on the centromere was less complete for CENPC1 compared with CENPA, INCENP, and survivin. A similar reduction in CENPC1 signal at the centromere was observed following RNaseA treatment and immunofluorescence analysis performed using a different anti-CENPC1 antibody provided by Dr. Bill Earnshaw (data not shown). (i) Merged images of ii and iii.
