Figure 6.
Deletion analysis and regulatory control of nucleostemin. (A) Schematic diagram (left) and intracellular distribution (right) of mutant nucleostemins lacking individual structural domains. (B) Overexpressing mutant nucleostemin decreases the number of cells in prophase (left, revealed by p-H3 staining), particularly with the dG4 and dG1 mutants, but not the number of cells in S phase (middle) in U2OS cells. Statistical analyses were carried out by scoring randomly chosen HPFs from two independent triplicate experiments (n = 6). (Right) BrdU-GFP immunofluorescence shows the patterns of BrdU labeling in wild-type or mutants-transfected cells during S phase. (C) Overexpressing dG4, dG1, and, to a lesser extent, dB/G1 mutant nucleostemin increases the number of apoptotic cells in U2OS cells (left) but not in Saos-2 cells (right). Statistical analyses were carried out by scoring randomly chosen HPFs from two independent triplicate experiments (n = 6). (D). The expression levels of p53 and ARF in CNS stem cells and cultures treated with 10% serum for 2 d and 14 d were measured by semiquantitative RT–PCR. 18S rRNA with 2:8 primer to Competimer ratio was used as a positive control; RT(−) reaction, carried out without RT in the first-strand synthesis reaction, was used as a negative control. Amplication cycles are p53 (29×), ARF (35×), 18s (23×), and RT(−) (33×). (E). Nucleostemin and p53 exist in a protein complex. In GST pull-down assays, p53 is specifically retained in the GST-raNS fraction and later detected by α-nucleostemin (2438) Western blot analysis (left, top) and vice versa (left, bottom). Such interaction can also be shown by coimmunoprecipitation of endogenous p53 and nucleostemin (right) in HEK293 cell lysate by α-nucleostemin (1164) antiserum but not by preimmune serum (Pre-Im). (F). Mapping of p53-interacting region in nucleostemin by GST pull-down assay. Same amount of GST-p53 and comparable amount of HA-tagged nucleostemin proteins (with the exception of dG4 and dG1) was used in each reaction. The retained proteins (bound) were detected by α-HA Western blot and expressed as ratios to the input. Error bars, S.E.M. Bars: A,B, 10 μm.