FIG. 4.

Overexpression of NS activates p53, induces G₁ arrest, and inhibits cell proliferation. (A) Overexpression of NS induces and activates p53. U2OS cells were transfected with the GFP, GFP-NS, GFP-NS^G1dm, or GFP-NS^ct plasmid. The GFP-expressing cells were then sorted by FACS into GFP dim-positive (GFP⁺) and GFP bright-positive (GFP⁺⁺) populations. Gating scales are shown in Fig. S2B in the supplemental material. The cell lysates were assayed by IB for the expression of p53, MDM2, p21, and GFP-NS. (B) Overexpression of NS induces G₁ arrest. U2OS cells were transfected with GFP, GFP-NS, GFP-NS^G1dm, or GFP-NS^ct. Forty-eight hours posttransfection, the GFP-expressing cells were gated for cell cycle analysis. The mean percentage of cells in G₁ phase is shown. The histograms of PI staining from one representative experiment are shown in Fig. S2C in the supplemental material. Asterisk, P < 0.01, compared to that of GFP. (C and D). Overexpression of NS inhibits cell proliferation, as determined by colony formation assay. U2OS cells were transfected with the GFP, GFP-NS, GFP-NS^G1dm, or GFP-NS^ct plasmid and sorted into GFP⁺ and GFP⁺⁺ populations as described for panel A. Equal numbers of cells were plated and grown in media containing G418 for 3 to 4 weeks. The average numbers of colonies are shown in panel D.