FIG. 7.

Depletion of MDMX and/or MDM2 by siRNA increases the protein level of p21 and causes G₁/early S-phase accumulation. (A) siRNA specifically for human MDMX (siMDMX) inhibits MDMX expression and induces p21 in p53-null H1299 cells. H1299 cells were transfected with 300 nM of a scrambled RNA duplex or the anti-MDMX siRNA duplex and harvested 48 h posttransfection. The cell lysates (100 μg/sample) were used to detect MDMX, p21, and tubulin (loading control) by WB, as indicated. (B) Depletion of MDMX by siRNA results in G₁ cell accumulation. H1299 cells were transfected as indicated and treated with 150 nM nocodazole for 16 h before harvest. The cells were harvested at 40 h posttransfection and analyzed for DNA content. A total of 50,000 cells were counted for each sample. The error bars represent the standard deviation of results from three independent experiments. Data were analyzed for statistical significance using the Student's t test.*, P < 0.05. (C) Further knockout of MDMX in p53 and MDM2-double-knockout MEF cells leads to more G₁ arrest. The cultured p53^−/− p53^−/− and MDM2^−/− or p53^−/− MDM2^−/− and MDMX^−/− MEF cells (Barboza et al., submitted) were treated with 150 nM nocodazole for 16 h. The cells were harvested analyzed for DNA content. A total of 50,000 cells were counted for each sample. (D to G) Depletion of MDMX or/and MDM2 by siRNA causes the accumulation of cells in G₁/early S phase accompanied by increased p21 levels. The H1299 cells transfected with scramble siRNA, MDMX siRNA, or MDM2 siRNA were synchronized by a double-thymidine arrest and harvested 0, 2, 4, 6, and 8 h after the second release. The cell pellets were split into two aliquots: one for the cell cycle by FACS analysis (C to E) and the other for WB to determine protein levels (see Fig. S6 in the supplemental material). c, nonsynchronized cells used as a control in both WB and FACS analyses. (H) Depletion of p21 by siRNA (sip21) rescues cells from G₁ arrest caused by depletion of MDM2 and MDMX. H1299 cells were transfected as indicated and treated with 150 nM nocodazole for 16 h before harvest. The cells were harvested at 40 h posttransfection and analyzed for DNA content. A total of 50,000 cells were counted for each sample. siMX-M2, MDMX and MDM2 siRNAs. The error bars represent the standard deviations of results from three independent experiments. Data were analyzed for statistical significance using Student's t test. *, P < 0.05. (I) Model for MDMX/MDM2-mediated p21 proteasomal turnover during the cell cycle (see Discussion for details).