Supplementary Figure 4. Nutlin-3 Disrupts the Binding Mdm2 to P73 in P53 Wild-Type Cells

Supplementary figure 1. Down-regulation of MdmX does not correlate with apoptotic response to Nutlin-3. (A) Cells were treated with 10 M of Nutlin-3 for 48 hrs. For vehicle controls, equal volumes of DMSO were added. Cells were harvested and the percentage of Annexin V positive cells was determined. (B) Cells were treated with 5 or 10 M of Nutlin-3 for 24 hrs. Cells were harvested and total lysates were subjected to immunoblot analysis for MdmX and Mdm2. (C) HCT116 cells were transfected with non-silencing siRNA (control) or MdmX siRNA. 24 hrs after transfection, cells were treated with 5 or 10 M of Nutlin-3for 72 hrs. Cells were harvested and the percentage of Annexin V positive cells was determined (left panel). Total lysates were subjected to immunoblot analysis for MdmX (right panel).

Supplementary figure 2. Arf does not affect apoptotic response to Nutlin-3 in SJSA-1 cells. (A) SJSA-1 cells were transfected with 100 nM of non-silencing siRNA (Control) or 100 nM of Arf siRNAs (Arf-1 and Arf-2 siRNAs are against p14ARF-specific exons, CDKN2A siRNA is against p16INK4A and p14ARF). 3 hrs after transfection, cells were treated with indicated amounts of Nutlin-3 for 72 hrs. Cells were harvested and total lysates were subjected to immunoblot analysis for p14ARF and p16INK4A (right panel). (B) NIH3T3cells transduced with a control-ER retrovirus or a retrovirus expressing E1A-ER, Myc-ER, Akt-ER or Raf-AR were treated with 200 nM of 4-HT or equal volumes of EtOH together with 20 M of Nutlin-3 for 48 hrs. Caspase-3/7 activity was measured and normalized to a number of viable cells, and indicated as a fold induction compared to EtOH control upon Nutlin-3 treatment. The average from three independent experiments is shown with standard deviation.

Supplementary figure 3. E2F transcriptional activity is higher in the highly sensitive cell lines than the low sensitive cell lines. Cells were co-transfected with E2F-Luc reporter plasmid together with pRL-TK plasmid as an internal control. 24 hrs after transfection, cells were treated with 5 or 10 M of Nutlin-3 for 24 hrs. Then, cells were harvested and luciferase activity was determined. Average of luciferase activities from three independent experiments is shown with standard deviation.

Supplementary figure 4. Nutlin-3 disrupts the binding Mdm2 to p73 in p53 wild-type cells.

Y-79 or DU4475 cells were treated with 10 M of Nutlin-3 for 6 hrs. Then, total cell lysates were prepared and equal amounts of cell extracts were subjected to immunoprecipitation analysis with agarose-conjugated anti-Mdm2 antibody. For control, equal amount of agarose-conjugated mouse IgG were used for immunoprecipitation. Bound proteins were separated and subjected to immunoblot analysis for Mdm2 and p73.