1. Supplementary Figures
A375, treated for 6 hours
DMSO
Nutlin-3
(5μM)
RG7388
(1μM)
2.5
MDM2 antagonists
WIP1i(μM)
WM35, treated for 6 hours
DMSO
Nutlin-3
(5μM)
RG7388
(1μM)
2.5
C8161, treated for 6 hours
DMSO
Nutlin-3
(5μM)
RG7388
(1μM)
2.5
Wip1
p53
Phospho-p53(Ser15)
Acetyl-p53(Lys 382)
MDM2
p21
GAPDH B
A375
6 hr
24 hr
HDM201 (nM) 20
100
Wip1i (μM)
2.5
C8161
6 hr
24 hr 20
100
2.5
WIP1
p53
P-p53(Ser15)
Acetyl-p53(Lys 382)
MDM2
p21
GAPDH C
Supplementary Figure S1. Immunoblotting and Reverse Phase Protein Array (RPPA) analysis of p53WT cells after treatment with MDM2 antagonist and GSK (WIP1i). (A) A375, WM35 and C8161 cells treated with 5 µM nutlin-3 or 1 µM RG µM WIP1i for 6 hours. (B) A375 and C8161 cells treated with HDM201 + µM WIP1i for 6, 24 hours. (C) Expression of phosphorylated-p53 (Ser 15) detected by protein array in response either 5 μM Nutlin-3, 1 μM RG7388 or combinations with 2.5μM WIP1i for 6 hours relative to DMSO solvent and tubulin control in A375 cells. Alkaline phosphatase (AP) was added to lysates after combination treatment as a control for specificity of the antibody. Significance of differences (*, p < 0.05) are indicated immediately above the bars for each treatment compared with DMSO control. The significance of differences for MDM2 antagonist treatment with or without WIP1i are also indicated. Data are presented as mean ± standard error of mean (SEM) for three independent repeats.

2. A C B D
p=0.10 ** * ** **
p=0.11
Supplementary Figure S2. GSK (WIP1i) potentiated the growth inhibitory and cytotoxic activity of HDM201. (A) The growth inhibition measured by SRB assay for p53WT (A375, WM35 and C8161) and p53MUT (WM164, WM35-R, CHL-1) melanoma cell lines treated with different concentrations of HDM201 combined with or without WIP1i (2.5µM) for 72 hours. (B) Summary of GI50 values (mean + SEM) for HDM201 with or without WIP1i in p53WT melanoma cells. (C) Clonogenic survival of P53WT melanoma cell lines treated with different concentrations of HDM201 combined with or without WIP1i (2.5µM) for 72 hours. (D) Summary of LC50 values for HDM201 from at least three independent repeats. SEM, standard error of the mean. *, p < 0.05; **, p <0.005.

3. C8161
Time (hrs) 1 2 4 6 8 24
RG μM - +
Wip1i 2.5 μM
WIP1
p53
Phospho-p53(Ser15)
Acetyl-p53(Lys382)
MDM2
p21
BAX
GAPDH
Supplementary Figure S3. Immunoblotting of C8161 cells treated with 0.2µM RG7388 with or without 2.5µM WIP1i for the indicated time.

4. Supplementary Figure S4
Supplementary Figure S4. ATM inhibitor (ATMi, KU55933) decreased the growth inhibition of A375 cells by MDM2 inhibitors. Summary of GI50 values (mean + SEM) for MDM2 inhibitors with or without ATMi in p53WT melanoma cells. *, p < 0.05, n.s., p > 0.05, SEM, standard error of the mean.
Forward
Reverse
WM35
Forward
Reverse
WM35-R
Supplementary Figure S5. Sanger sequencing of WM35 and WM35-R. Sanger sequencing showing WM35-R has a homozygous point missense mutation (1001G>T) resulting in a Gly334Val substitution in the p53 protein.

5. A
WM35
WM35-R
Nutlin-3 1μM - +
RG μM
Wip1i 2.5μM
WIP1
p53
Phospho-p53(Ser15)
Acetyl-p53(Lys382)
MDM2
p21
BAX
GAPDH B
WM35
RG7388
0.2μM
CHX 100μg/ml
Hour(s) after RG7388 4 6 8
Hour(s) after CHX 2
WIP1i 2.5 μM - +
WM35-R
RG7388
0.2μM
CHX 100μg/ml 4 6 8 2 - +
p53
Phospho-p53(Ser15)
Acetyl-p53(Lys382)
WIP1
MDM2
p21
GAPDH
Supplementary Figure S6. WM35 and WM35-R isogenic paired p53 wild-type and mutant cell lines demonstrated WIP1i potentiates MDM2 antagonists in p53-dependent manner. (A) Immunoblotting of paired WM35, WM35R cell lines treated by nutlin-3 (1μM), RG7388 (0.2μM) with or without WIP1i (2.5μM) for 6 hours. (B) Immunoblotting of WM35 and WM35R cells treated by RG7388 (0.2μM) followed by cycloheximide (CHX) 100μg/ml with or without WIP1i (2.5μM) .

6. A375
C8161
WM35
WM35-R
MDM2
MDM2
PUMA(BBC3)
CDKN1A
CDKN1A
PUMA (BBC3)
TP53INP1
TP53INP1
FAS
MDM2
FAS
TNFRSF10B
FAS
CDKN1A
BAX
TNFRSF10B
PUMA(BBC3)
TNFRSF10B
PUMA(BBC3)
TP53INP1
MDM2
TP53INP1
TNFRSF10B
BAX
BAX
CDKN1A
FAS
BAX
Supplementary Figure S7. Summary of p53 regulated gene induction by combination of 0.2 µM RG7388 and 2.5 µM GSK for 6 hours relative to DMSO solvent control. Summary data are presented as a combination of three independent repeats.
Supplementary Figure S8. MDM2, CDKN1A, TP53INP1, TNFRSF10B, PUMA(BBC3)
gene expression changes induced by RG7388 doses of 0.04, 0.2, and 1 µM, and combination of 0.2 µM RG7388 plus 2.5 µM GSK for 6 hours relative to DMSO solvent control. The dose dependent increase in transcripts by RG7388 was saturated at 0.2µM and no further increase was seen with the addition of 2.5µM WIPi.

7. A
DMSO
WIP1i
RG7388
RG WIP1i
HDM201
HDM201 + WIP1i B
Supplementary Figure S9. The effect of MDM2 and WIP1 inhibitor combination on cell cycle distribution and apoptosis for 48hr treatment. Melanoma cells were treated by either 2.5μM WIP1i, 0.2 μM RG7388, 0.2 μM HDM201 or combinations for 48 hours. (A) Cell cycle distribution changes measured by FACS. (B) Sub-G1 events by FACS as a measure of apoptosis. Statistically significant p-value (*, p < 0.05) was shown. Data are presented as mean ± standard error of mean (SEM) of three independent repeats.

8. A
DMSO
WIP1i
RG7388
RG WIP1i
HDM201
HDM201 + WIP1i B
Supplementary Figure S10. The effect of 72hours treatment with combinations of MDM2 and WIP1 inhibitors on cell cycle distribution and apoptosis. Melanoma cells were treated by either 2.5μM WIP1i, 0.2 μM RG7388, 0.2 μM HDM201 or combinations for 72 hours. (A) Cell cycle distribution changes measured by FACS. (B) Sub-G1 events detected by FACS as a measure of apoptosis. Statistically significant p-value (*, p < 0.05) was shown. Data are presented as mean ± standard error of mean (SEM) of three independent repeats

9. JEG-3
MRK
HCT116 p53+/+
HCT116 p53-/-
A375
WM35
CHL-1
C8161
MDMX
WIP1
GAPDH 98 64 50
Supplementary Figure S11. Immunoblotting of basal expressions of MDMX and WIP1 in a panel of cells