Figure 5. Effects on cell number and p53 activation in IMCD cells exposed to H₂O₂ depending upon the reduced levels of TRIP13.

(A,B) Representative data of TRIP13 protein (A) and Trip13 mRNA (B) using immunoblot and quantitative RT‐PCR analysis, respectively, from IMCD-3 cells transduced with either Trip13 cDNA (Trip), control short hairpin RNA (Csh) or Trip13-specific shRNA (B6). Arrows in (A) indicate protein size marker. β‐actin was used as a loading control for the immunoblot analysis, and 18S was used as a normalization control for the quantitative RT-PCR analysis. (C) Cell number analysis. IMCD cells expressing control shRNA (Csh) or Trip13-specific shRNA (B6) were plated and counted over a 72 hour period by hemocytometry. n = 3 different experiments performed in triplicate per time point and group. **P < 0.01 significant difference between groups. (D–H) Immunoblot analyses of various post-translational changes to p53 normalized to total p53 levels following 8.8 μM hydrogen peroxide (H₂O₂) versus vehicle (veh) treatment in Csh and B6 IMCD cells. Quantification of (E) phospho-p53 at Serine 15, (F) acetylated p-53 at Lysine 382, (G) phospho-p53 at Serine 33, and (H) phospho-p53 at Serine 37 were analyzed from 3 independent experiments. *P < 0.05 significant difference between B6 and Csh cells following H₂O₂ treatment.