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).

F395C QC WT Q392G C398L QQ

EVHCT116 p53+/+ p<0.01 p<0.01 p<0.01 p<0.01 p<0.01 p<0.01

HCT116 p53-/- p<0.05 p<0.05 p<0.05 p<0.05 p<0.05 p<0.05

WTHCT116 p53+/+ p<0.01 p<0.01 p=0.83 p=0.88 p=0.35

HCT116 p53-/- p=0.67 p=0.91 p=0.49 p=0.79 p=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.

F395C QC WT Q392G C398L QQ

EVHCT116 p53+/+ p<0.01 p<0.01 p<0.01 p<0.01 p<0.01 p<0.01

HCT116 p53-/- p<0.01 p<0.01 p<0.01 p<0.01 p<0.01 p<0.01

WTHCT116 p53+/+ p<0.01 p<0.01 p=0.24 p=0.18 p=0.52

HCT116 p53-/- p=0.08 p=0.39 p=0.62 p=0.98 p=0.50

B

F395C QC WT Q392G C398L QQ

EVWT p<0.01 p<0.01 p<0.01 p<0.01 p<0.01 p<0.01

T55D p<0.01 p<0.01 p<0.01 p<0.01 p<0.01 p<0.01

WTWT p<0.01 p<0.01 p=0.04 p=0.32 p=0.34

T55D p=0.81 p=0.74 p=0.04 p=0.33 p=0.34

C

B56

γ3

A38

3G

EV

p53

p21

Thr55 P

vinc

HA

F39

5C

Input HA-IP

vinc

PP2A A

p53

PP2A C

HA

B56

γ3

A38

3G

EV

B56

γ3

A38

3G

EV

376-IHGLIYNALKLFMEMNQKLFDDCTQQF-402 G C

B56γ: Cancer:

B C

D

A

p53

vinc

B56γ3

HCT116 +/+

B56

γ3

A38

3G

M

HCT116 -/-

B56

γ3

A38

3G

M

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

% C

ells

at 1

20 h

ours

EA383G WT

EVHCT116 p53+/+ p<0.01 p<0.01

HCT116 p53-/- p<0.01 p<0.01

WTHCT116 p53+/+ p<0.01

HCT116 p53-/- p=0.85