RNaseOne treatment and in situ “rescue” assay. Cells cytospun on slides were permeabilized with Triton X-100 and subjected to RNaseOne treatment for 20 min (this treatment resulted in the significant delocalization of both CENPC1 and INCENP from the kinetochores; see Fig. 6) followed by fixation with 4% formaldehyde. Cells were then incubated with RNase inhibitor (RNasin) and in vitro transcribed α-satellite RNA for 1 h at 37°C, before overnight incubation with the relevant myc-tagged CENPC1 and INCENP recombinant proteins. The recruitment of these proteins at the kinetochore (detected with CREST antibody) and nucleolus was detected using antibody against the myc tag. (A–D) Effects on the nucleolus. The “mean ratio” of the immunofluorescence signals of CENPC1-myc (Aiii,Biii; green) and INCENP-myc (Ciii,Diii; green) normalized against nucleophosmin-1 (NP-1) (Aii–Dii; red) was determined from the quantitative immunofluorescence scoring of the nucleoli from 25 cells, with (“rescued”) and without (“non-rescued”) replenishment using in vitro transcribed α-satellite (α13/21) RNA. The RNA replenishment led to a significant enrichment (P-value < 0.001) of CENPC1-myc (Aiii,Biii; green) and INCENP-myc (Ciii,Diii; green) staining in the nucleoli of most cells. (E–H) Effects on the mitotic centromere. The “mean ratio” of the immunofluorescence signals of CENPC1-myc and INCENP-myc normalized against the CREST signals (Eii–Hii; green) for both “rescued” (using α13/21) and “non-rescued” cells was determined from the quantitative immunofluorescence scoring of 50 chromosomes. The α-satellite RNA replenishment resulted in a modest recovery in the binding of CENPC1-myc and INCENP-myc seen only at the kinetochores of some of the chromosomes.
