Fig. 3
ORF1p nuclear localization in 2102Ep cells is not strictly influenced by cell cycle status. a Cell cycle arrest was induced for 22 hours with 10 μg/ml aphidicolin or 3 mM hydroxyurea and confirmed by propidium iodide staining and flow cytometry. The percentages of cells in G0/G1, S or G2/M phases were determined using BD CELLQuest software (BD Biosciences). FL2-Area is plotted against cell counts. b Percentage of cells having visible nucleolar localization when not treated (NT) or treated with aphidicolin to induce cell cycle arrest at G0/G1 phase. c Endogenous ORF1p was detected by Western blotting in both nuclear and cytoplasmic cell fractions left untreated (NT) or treated with aphidicolin (APH) or hydroyxurea (HU). Purities of nuclear and cytoplasmic fractions are shown using α-Lamin A/C and α-MEK1/2 antibodies, respectively. WCL: whole cell lysate. d,e) Immunofluorescence of 2102Ep cells showing that cells both with or without nucleolar ORF1p localization can express CDT1 or Geminin. f The percentages of untreated 2102Ep cells having visible ORF1p nucleolar localization that are marked (+) or unmarked (-) by α-CDT1 (red bars) or Geminin (green bars) staining. The data summarizes three replicate experiments with at least 400 cells scored for each experiment. Statistical significance was calculated by Student’s t-test (** p<0.01; *** p<0.001)