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. 2013 May 29;288(29):21307–21319. doi: 10.1074/jbc.M112.445890

FIGURE 6.

FIGURE 6.

p53-induced down-regulation of RUNX2 is independent of cell cycle inhibition. A, to address whether Nutlin-3-induced RUNX2 down-regulation is a consequence of a p53-p21 pathway-induced cell cycle block, U2OS cells were transfected with non-silencing siRNA (NS) or siRNA against p21 (si-p21) for 24 h before Nutlin-3 treatment. Nutlin-3 treatment results in dose-dependent reduction in cell growth in non-silencing siRNA-treated cells. Silencing p21 by siRNA rescues Nutlin-3-induced growth defect. B, to investigate the cell cycle progression, flow cytometry analysis was performed after 48 h of Nutlin-3 treatment. Knockdown of p21 rescues the fraction of cells in S-phase compared with non-silencing siRNA treated cells (34 and 19%, respectively). C, Western blot analysis reveals a marked increase in p53, p21, and MDM2 levels and down-regulation of RUNX2 in non-silencing siRNA-transfected cells as response to Nutlin-3. Although si-p21 effectively reduces p21 protein levels, Nutlin-3 treatment results in drastic up-regulation of p53 and down-regulation of RUNX2. *, p < 0.05.