FIGURE 8.

NPM1 is required for nucleolar tethering of HP1γ-enriched foci. A, U2OS cells were transfected singly with plasmids encoding EGFP alone, EGFP-HP1α, EGFP-HP1β, EGFP-HP1γ, and EGFP-HP1γ-mut (C59R) as indicated. Twenty-four hours after transfection, cell lysates were prepared, and co-IP was conducted with a rabbit anti-GFP antibody. The precipitates were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and blotted with a mouse anti-NPM1 antibody or a mouse anti-GFP antibody, respectively. Approximately 5% of the cell lysate is shown as loading control. IP, immunoprecipitation. B, U2OS cells were transfected with plasmids encoding wild type EGFP-HP1γ or EGFP-HP1γ mutant. After 2 days (48 h) the cells were fixed, immunofluorescence-stained for NPM1 (red), and overlaid with the green GFP signal. WT EGFP-HP1γ, but not the C59R mutant, co-localized with NPM1 in the perinucleolar regions as indicated by arrowheads. C, p53^−/−,Npm1^−/− MEFs were transfected with plasmids encoding EGFP-HP1γ, Myc-NPM1, or empty pCDNA3 vector. Cells were stained for NPM1 (red) and fibrillarin (blue), which were merged with the green EGFP signal. D, cells from the experiment shown in C were evaluated for perinucleolar association of rounded HP1γ foci. Shown are the percentages of WT, p53^−/−, and p53^−/−,Npm1^−/− cells that displayed nucleolus-associated round HP1γ foci. Data were determined from three independent experiments, and 200 cells were evaluated for EGFP-HP1γ and EGFP-HP1γ + Myc-NPM1 plasmid combinations, respectively. Shown is mean ± S.D. *, p < 0.05.