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Fig. 4. Localization of GFP-NoLS in HeLa cells during the cell cycle. (A) HeLa cells were transfected with GFP-NoLS fusion and analyzed by fibrillarin or B23 immunolabeling (a,e), GFP-NoLS observation (b,f) and phase-contrast (d,h). The nucleolar localization of NoLS in interphasic cells was demonstrated using fibrillarin, a marker of the DFC, and B23 preferentially localized to the GC of nucleoli. GFP-NoLS did not colocalize with fibrillarin as observed by comparing the enlargements (a-c, insets), whereas it colocalized with B23 as observed by comparing the enlargements (e-g, insets). (B) HeLa cells cotransfected with GFP-NoLS and DsRed-B23 were synchronized in mitosis. GFP-NoLS observations (a,e,i), DsRed-B23 observations (b,f,j) and superimpositions of both labelings in the same mitotic cells (c,g,k). The mitotic stage was determined by DAPI staining (d,h,l). GFP-NoLS colocalized with DsRed-B23 at the chromosome periphery in metaphase (a-d) and in numerous foci, the PNBs, in telophase (e-h) and early G1 (i-l). In early G1, GFP-NoLS and DsRed-B23 colocalized in the reformed nucleoli. These nucleoli are indicated by arrowheads (i-k). Bars, 10 µm.
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