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Supplimental figure 1. Melanoma free survival curve (A) and summary table (B) for median survival and melanoma free survival for each genotype. All of.

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Presentation on theme: "Supplimental figure 1. Melanoma free survival curve (A) and summary table (B) for median survival and melanoma free survival for each genotype. All of."— Presentation transcript:

1 Supplimental figure 1. Melanoma free survival curve (A) and summary table (B) for median survival and melanoma free survival for each genotype. All of them are Ink/Arf-/-. B. A.

2 AL4 Supplimental figure 2. AL4 melanoma cell (RAS-Ink4a/Arf) shows increased invasion (A, top: quantitation, bottom: images) when Pten expression is downregulated with shRNA targeting Pten, which correlates with decreased E-Cadherin expression both at the protein level analyzed by western blot (B) and mRNA level addressed by semi-quantitative rt-PCR analysis (C). ECAD NCAD pSTAT PTEN pERK pFAK pAKT EVSh4Sh11 Cyclin D1 Actin A.B. R15 EV-Sh4Sh11 Pten ECAD C.

3 Supplemental Figure 3. Loss of Pten expression with Sh4 in AL4 melanoma cell caused increased AKT2 phosphorylation compared to control with EV. (A) AKT (Pan), AKT1, AKT2, and AKT3 was immunoprecipitated with each antibodies (all except for AKT3 (Upstate) was from Cell signaling technology) and was blotted with p-AKT antibody and then with Akt antibody. (B) Quantitation by ImageJ showed 1.9 fold increase of active AKT2 (pAKT2/AKT2 immunoprecipitated) in cells with Sh4 compared to cells with EV. AKT P-AKT WB: Pan123AKT IP:Pan123 Sh 4EV A.B. pAKT/AKTEVSh4 AKT11.321.46 AKT20.180.35 AKT31.331.19 Ratio of pAKT/AKT in AL4 with Sh4 vs. EV

4 Ctrl myrAKT3 pAKT pSTAT pFAK PTEN Ctrl myrAKT3 CN116 Ponceau Invasion (10 6 pixels/well) CN44 AKT2 Actin A.B. Supplemental figure 4. (A) Further increase of AKT3 activity in CN116 cells by ectopic expression of myristoylated AKT3 can not enhance invasion compared to empty vector control. pBabe-puro-myr-HA-AKT3 (Addgene Plasmid # 9019) were provided by Dr. William Sellers. (B) Dominant negative AKT2 expression in CN44 cells decreases invasion compared to control cells expressing GFP protein. GFP dnAKT2

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