1. Exogenous B56γ3 WT F395C WT F395C IgG Vinc Endogenous B56γ2,3 HA-IPInput Figure S1 HA-B56γ3 is unable to interact with endogenous B56γ. Lysates of U2OS cells transfected with either wild type or F395C mutant B56γ3, were immunoprecipitated with HA antibody, then analyzed by western blot against B56γ and vinculin (vinc).

2. F395CQCWTQ392GC398LQQ EV HCT116 p53+/+p<0.01 HCT116 p53-/-p<0.05 WT HCT116 p53+/+p<0.01 p=0.83p=0.88p=0.35 HCT116 p53-/-p=0.67p=0.91p=0.49p=0.79p=0.72 A Figure S2 Statistical analysis of cell growth and transformation data. P-values for anchorage independent cell growth experiments from Figure 3 (A); for HCT116 cells growth experiments from Figure 4A (B); and for H1299 cell cells growth experiments from Figure 4B (C) were calculated by comparing the effect of expressing the various mutants either versus the EV control or versus the wild type protein. F395CQCWTQ392GC398LQQ EV HCT116 p53+/+p<0.01 HCT116 p53-/-p<0.01 WT HCT116 p53+/+p<0.01 p=0.24p=0.18p=0.52 HCT116 p53-/-p=0.08p=0.39p=0.62p=0.98p=0.50 B F395CQCWTQ392GC398LQQ EV WTp<0.01 T55Dp<0.01 WT p<0.01 p=0.04p=0.32p=0.34 T55Dp=0.81p=0.74p=0.04p=0.33p=0.34 C

3. B56γ3 A383G EV p53 p21 Thr55 P vinc HA F395C Input HA-IP vinc PP2A A p53 PP2A C HA B56γ3 A383G EV B56γ3 A383G EV 376-IHGLIYNALKLFMEMNQKLFDDCTQQF-402 G C B56γ: Cancer: BC D A p53 vinc B56γ3 HCT116 +/+ B56γ3A383G M HCT116 -/- B56γ3A383GM Figure S3 The A383G mutant is unable to promote p53-dependent tumor suppressive functions. (A) Amino acid sequence of B56γ showing the p53-interaction domain, adjacent regions and the cancer-derived mutations. Lysates of U2OS cells transfected with HA-tagged B56γ3, A383G, or an empty vector control, were either immunoprecipitated with anti-HA antibody, then analyzed by western blot against p53, PP2A A and C, HA, and vinculin (B); or analyzed for p53 Thr55 phosphorylation, p21 protein levels, HA, p53, and vinculin (C). (D) Representatives of cell proliferation of HCT116 cells were transfected with either wild type, or A383G, or an empty vector control. Numbers of cells present at the 120 hour time point were normalized against the representative empty vector controls and plotted in a bar graph. Error bars show average +/- s.d. from triplicate plates in one representative experiment. Cell lysates were analyzed by immunoblotting of the transfected HA-B56γ3, endogenous B56γ3/B56γ2, and p53 proteins. M: empty vector-transfected cell lysate at seeding. (E) Statistical analysis of cell growth from (D). EV WT A383G HCT116 +/+HCT116 -/- 0 120 80 60 40 20 100 % Cells at 120 hours E A383GWT EV HCT116 p53+/+p<0.01 HCT116 p53-/-p<0.01 WT HCT116 p53+/+p<0.01 HCT116 p53-/-p=0.85