Fig 2: Upregulation of p53 and p53-target proteins upon HDM2 inhibition. Cells were exposed to increasing concentrations of HDM2 SMIs for 24 h. Whole cell lysates were subjected to SDS-PAGE and probed for specific proteins. Western blots show changes in p53 and its target proteins after 24 h for the 4 cell lines studied. A) The two wt-p53 cell lines, WSU-FSCCL (A1) and KM-H2 (A2) are presented in the top panel. B) The two mt-p53 cell lines, WSU-DLCL2 (B1) and RL (B2) are presented in the lower panel. Representative blots are shown.

Fig 3: Biological response of lymphoma cell lines to HDM2 SMIs. Nutlin-3 (Red bars) and MI-219 (Blue bars) represent cell viability for 4 cell lines exposed to increasing concentrations of HDM2 SMIs for up to 72 h. Comparison for overall differences across equivalent concentrations were significant between the two HDM2 SMIs (ANOVA; p = 0.001) for each time period (24 to 72 h) in both wt-p53 cell lines. Numerical values located under the graphs represent percentage of Annexin V positive cells for Nutlin-3 (red print) and MI-219 (blue print). MI-219- induced increases in Annexin V positive WSU-FSCCL cells at 24 and 48 h was significantly greater than that for Nutlin-3 (p = 0.0001) within the same cell line. For KM-H2 cells, the difference was significant only at 24 h (p = 0.026). Columns and error bars represent Mean ± S.D. of three independent experiments.

Fig 4: Effect of HDM2 inhibition on p53-dependent gene expression. WSU-FSCCL cells were exposed to increasing concentration of Nutlin-3 and MI-219 for 12, 24 and 48 h. Baseline gene expression and after treatment were quantified by qRT-PCR relative to GAPDH using the ??Ct method and expressed as fold induction of gene expression relative to that in the untreated control. mRNA expression levels for p53 (A), HDM2 (B), p53AIP1 (C) and p21 (D). Coded colors represent different concentrations and are listed for each HDM2 SMI at the base of the page. Error bars plotted represent mean values ± SE performed in triplicate from two independently treated experiments.

Fig 5: Effect of HDM2 inhibition in normal B-lymphocytes derived from healthy donors. A) Isolated B-lymphocytes from normal donors were exposed to HDM2 SMIs for up to 48 h. Neither HDM2 SMI induced a significant decrease in cell viability compared to untreated cells (control). B) The inactive Nutlin-3 analog, Nutlin-3b, was ineffective in significantly decreasing cell viability in all 4 cell lines for up to 72 h. Columns and error bars represent Mean ± S.D. of three independent experiments.