FIGURE 1.

A, the diagram illustrates the growth conditions of 4pX-1 cells. 4pX-1 cells were serum-starved for 18 h (14) followed by the addition of 10% FCS for 10 h, expression of pX by tetracycline (5 μg/ml) removal for an additional 10 h, and nocodazole (250 ng/ml) treatment for the last 6 h of growth. Table 1, flow cytometric profiles of 4pX-1 cells grown according to the diagram in A. +10 h indicates growth for 10 h in 10% FCS; +20 h indicates growth for 20 h in 10% FCS with 10-h expression of pX; +26 indicates growth for 26 h in 10% FCS, 16 h growth with pX expression, and 6 h with nocodazole treatment. Quantification is from at least three independent flow cytometric experiments. Histogram is the quantification by flow cytometry of 4pX-1 cells containing >4N DNA, grown with (+) or without (-) pX expression by tetracycline removal, as represented in the diagram; nocodazole (250 ng/ml) was added for the last 6 h; SB202190 (5 μm) was added for the last 16 h. Results are from at least three independent experiments. B, flow cytometric profiles of 4pX-1 cells grown as in Fig. 1A, ±pX, sorted for cells containing 2N, 4N, and >4N DNA. The circled area indicates 4pX-1 cells with >4N DNA. AU, absorbance units. C, Hoechst staining of sorted 4pX-1 cells containing 2N, 4N, and >4N DNA. Nuclear size of sorted cells quantified from ∼500 cells, employing Image J software. Results are from three independent experiments. D, Western blot analysis of p53 in WCE isolated from 4pX-1 cells, grown as in Fig. 1A with (+) or without (-) pX and with (+) or without (-) nocodazole (250 ng/ml).