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 GSK2830371 (WIP1i). (A) A375, WM35 and C8161 cells treated with 5 µM nutlin-3 or 1 µM RG7388 + 2.5 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.

A C B D p=0.10 ** * ** ** p=0.11 Supplementary Figure S2. GSK2830371(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.

C8161 Time (hrs) 1 2 4 6 8 24 RG7388 0.2μM - + 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.

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.

A WM35 WM35-R Nutlin-3 1μM - + RG7388 0.2μM 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) .

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 GSK2830371 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 GSK2830371 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.

A DMSO WIP1i RG7388 RG7388 + 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.

A DMSO WIP1i RG7388 RG7388 + 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

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