Nucleolin mobilization does not occur in p53-null Saos-2 cells. Saos-2 and U2-OS cells were either left untreated (A and D, respectively), subjected to IR (10 Gy) (B and E), or exposed to heat shock (90 min at 44°C) (C and F). Cells were fixed by treatment with 4% (wt/vol) formaldehyde for 30 min at RT and then stained for nucleolin as described in Materials and Methods.

The C terminus of p53, in combination with cell stress, is required for nucleolin mobilization. H1299 cells expressing either p53-13 (A to D), p53-22/23 (E and F), or p53ΔC30 (G and H) were grown in the presence (A and B) or absence (C to H) of tetracycline. Cells were either treated with IR (10 Gy) (B, D, F, and H) or left nonirradiated (A, C, E, and G). Cells were fixed by treatment with 4% (wt/vol) formaldehyde for 30 min at RT and stained for nucleolin as described in Materials and Methods.

Selective nucleolin mobilization induced by IR, CPT treatment, or heat shock. U2-OS cells were either left untreated (A) or subjected to IR (10 Gy) (B), treatment with CPT (2 μM for 2 h) (C), heat shock (15 min at 44°C) (D), HU (2 mM for 1 h) (E), or UV (a single 30-J m⁻² treatment) (F). The kinetics of nucleolin relocalization were investigated in U2-OS cells by first exposing cells to 10 Gy of IR (G) and then examining the localization of nucleolin 20, 60, 120, or 180 min postirradiation (H through K, respectively). In all cases, cells were fixed by treatment with 4% (wt/vol) formaldehyde for 30 min at RT and stained for nucleolin as described in Materials and Methods. Nucleolin localization was observed by epifluorescence microscopy using a Zeiss Axiophot.

Nucleolin-p53 complex formation is selectively induced in response to stress. Cell lysates were prepared from U2-OS cells at various times (as indicated) after either exposure to 10 Gy of IR (A, B, and C), treatment with 0.5 μM CPT for 1 h (D), or UV irradiation with a single 30-J m⁻² dose (E). For cells treated with CPT, times indicate the amount of time elapsed after the beginning of treatment. Extracts (0.5 ml; 450 μg of protein) were subjected to immunoprecipitation (IP) with a mouse monoclonal antibody against nucleolin or total p53 or with a rabbit anti-(pSer15)p53 polyclonal antibody (as indicated). For panel C, where indicated, lysates were treated with ethidium bromide (EthBr) and RNase prior to immunoprecipitation. The precipitated material was subjected to SDS-PAGE and then to immunoblot analysis using an anti-nucleolin, anti-p53, or anti-β-actin antibody, as indicated to the left of each panel. Total cell lysates (20 μg) were electrophoresed to monitor the cellular amount of nucleolin, total p53, or (pSer15)p53 following stress.

(A through H) Selective nucleolin mobilization occurs in MRC-5 cells. Nonimmortal MRC-5 lung fibroblasts, either untreated (A to D) or 60 min after exposure to γ-irradiation (10 Gy) (E to H), were processed for immunofluorescence analysis. Subsequent to Triton X-100 extraction and fixation, cells were stained by using a combination of mouse anti-nucleolin monoclonal (A and E) and rabbit anti-UBF polyclonal (B and F) antibodies. Cells were then stained with secondary fluorescein isothiocyanate-labeled anti-mouse and Texas Red-labeled anti-rabbit antibodies and were examined by epifluorescence microscopy. Composite images of the combined staining patterns for nucleolin and UBF are shown (C and G), as are images of the 4′,6′-diamidino-2-phenylindole (DAPI)-stained nuclei (D and H). (I) Nucleolin-p53 complex formation in MRC-5 cells. Lysates (0.5 ml; ∼400 μg of protein) from control MRC-5 cells (lane 1) or from cells processed 20, 60, or 180 min after exposure of cells to 10 Gy of IR (lanes 2 to 4, respectively) were subjected to immunoprecipitation using a mouse anti-nucleolin monoclonal (top panel) or a rabbit anti-total p53 polyclonal (second panel) antibody. Immunoprecipitates (IP) were analyzed by Western blotting for the presence of total p53 (top panel) or nucleolin (second panel). Aliquots of the lysates (10 μg) were examined for the level of p53 (third panel) or nucleolin (bottom panel). (J) Lack of UBF-p53 complex formation in MRC-5 cells. A procedure similar to that for panel I was used, except that lysates were immunoprecipitated with p53 antibodies and precipitates were tested for the presence of UBF (top panel). Lysates were analyzed for the presence of UBF (second panel), total p53 (third panel), and actin (used as a loading control) (bottom panel).

The extreme C terminus of p53 is essential for nucleolin-p53 complex formation in vivo and in vitro. (A) Nucleolin was immunoprecipitated (IP) (upper panel) from cell extracts (0.5 ml) prepared from untreated (lanes 1, 3, and 5) or γ-irradiated (lanes 2, 4, and 6) cells expressing wild-type or mutant p53. The cell lines used were p53-13 (lanes 1 and 2), p53-22/23 (lanes 3 and 4), and p53ΔC30 (lanes 5 and 6). Following SDS-PAGE of the immunoprecipitates, the separated material was subjected to immunoblot analysis using anti-(pSer15)p53 antibodies. Lysates (20 μg) prepared from control and IR-exposed cells were electrophoresed to monitor levels of (pSer15)p53 (second panel), total p53 (third panel), and the RPA middle subunit (RPA2) (bottom panel) in the extracts. The level of RPA2, used in this experiment as the loading control, does not change in response to γ-irradiation (data not shown). (B) Far-Western analysis of the nucleolin-p53 interaction. Equivalent amounts (500 ng) of full-length p53 (FL), the p53 N-terminal domain (NT; amino acids 1 to 160), middle domain (MD; amino acids 160 to 320), and C-terminal region (CT; amino acids 320 to 393), and p53 lacking the C-terminal 30 amino acids (ΔC30), each containing an N-terminal GST tag, were separated by SDS-PAGE. GST alone was also electrophoresed as a control. Following transfer to a nitrocellulose membrane, the membrane was probed with purified full-length nucleolin (1 μg in 5 ml) (lanes 1 to 6). The binding of nucleolin was visualized by using a nucleolin antibody. To visualize GST-tagged proteins, the membrane was stripped and subjected to immunoblot analysis using a rabbit anti-GST antibody (lanes 7 to 12). (C) Schematic showing the results of the far-Western analysis.