Fig. 3. p53-mediated DEPTOR expression suppresses cell proliferation, survival, and cell cycle progression.

a Deletion of p53-binding site C on the DEPTOR promoter abrogates the increase in DEPTOR expression in a cell density-dependent manner. U2OS cells, with or without the C site, were seeded at different concentrations (4 × 10⁵, 8 × 10⁵, and 16 × 10⁵) in 100 mm dishes and grown for 24 h (top) or seeded at the same density and grown for the indicated time periods (bottom). Cells were then harvested and subjected to IB with the indicated Abs. b Deletion of p53-binding site C on the DEPTOR promoter accelerates cell proliferation. Cells with or without site C of the DEPTOR promoter were seeded in triplicate in 96-well plates and grown for various time periods, followed by ATPlite-based cell proliferation assay. Cell proliferation is expressed as the fold change compared with day 1 (mean ± S.E.M, n = 3; *p < 0.05). c Deletion of p53-binding site C on the DEPTOR promoter promotes clonogenic survival. Cells with or without site C of the DEPTOR promoter were seeded in triplicate in 60 mm dishes at 300 cells per dish and incubated for 14 days, followed by staining (left) and colony counting (right) (mean ± S.E.M, n = 3; ***p < 0.001). d, e Deletion of p53-binding site C on the DEPTOR promoter accelerates cell cycle progression. Cells were treated with 2 mM thymidine for 14 h and then released for 9 h. Next, cells were treated with thymidine for an additional 14 h and then incubated in fresh medium for different time periods. Cells were then harvested for flow cytometry d or IB with the indicated Abs e.