(A) Immunoblot analyses of PTEN in cell lysates from early-passage Ras-Ink4a/Arf melanoma. The corresponding Pten genotype is shown on top of the blot. Note the different level of Pten in AL4 (high), CN41 (intermediate), and CN44 (loss), which were used in B and C. (B) (Top) PCR analysis on genomic DNAs of early passage AL4, CN41, and CN44 melanoma cells of Pten (exon 5) and Ink4a allele. (Bottom) Semi-quantitative reverse transcription PCR analysis for Pten mRNA expression (20 cycles). Ribosomal protein R15 mRNA expression serves as an internal control (19 cycles). (C) Boyden Chamber assay. Photographs of a representative field of the stained membrane show highest level of invasion through the Matrigel by CN44 melanoma cells. (D) Optical quantitation of Boyden Chamber assay on Ras-Ink4a/Arf-Pten+/+ (n=4) and Ras-Ink4a/Arf-Pten+/- (n=5) early passage melanoma cell lines. Pten wt cells showed overall lower invasive capacity (OD560 mean= 0.2152 ± 0.02781, N=4) when compared to Pten heterozygous cells (OD560 mean= 0.4268 ± 0.1160, N=5).

(A) Molecular profile comparison of Ras-Ink4a/Arf Pten+/+ and Pten +/- early passage melanoma cell lysates by immunoblotting with antibodies labeled on left. The corresponding Pten genotype is shown on top of the blot. E-cadherin protein levels are lower on Pten +/- melanoma cells compared to on Pten +/+ cells. (B-D) Immunoblot analysis CN116 (B) and C140 (C) with EV, sh4, or sh11, and CN44 (D) cells with GFP or PTEN expression with antibodies indicated. (E) Real time rt-PCR analysis of Pten and E-Cadherin expression in CN116 cells with EV, Sh4, or Sh11. (F) Boyden chamber assay (left) of CN116 cells following siRNA-mediated E-Cadherin downregulation (siECAD) compared to non-targeting control (siNT) and western blot analysis (right).

(A) LY294002 treatment decreased p-AKT level of CN116 cells with Pten loss (sh11) (top, immunoblots with indicated antibodies), which correlates with invasive capability (bottom, Boyden chamber assay). Invasion was quantitated by measuring absorbance of crystal violet dye. Representative images of invasion chamber are shown.
(B) Immunoblot analysis of CN116 RAS Ink/Arf melanoma cells with EV, Sh4, or Sh11. Note much higher increase of pAKT2 compared to total pAKT by loss of Pten protein expression.
(C&D) PTEN expression was knocked down with siRNA targeting PTEN (siPTEN) in WM1366 and its effect on AKT phosphorylation (total and AKT2) was measured by immunoblotting (C). p-AKT2 level was analyzed by ELISA (D).

(A) Kaplan-Meier melanoma free survival analysis of Ras-Ink4a/Arf mice with wild type (n=28) or heterozygous (n=48) Pten allele. Non-melanoma tumors were censored from this analysis. Statistically significant differences were detected between the two cohorts (p=0.0002) (B) H&E (left), Tyrosinase (middle) and Dct (right) staining of primary melanoma (top panels) and lung metastasis (middle panels). Bottom panel shows H&E of a probable melanoma metastasis on a lymph node, based on cell morphology. (C) Real time reverse transcription PCR analysis for mRNA level of Tyr-Hras transgene and Tyr, Trp1 and Dct melanocytic markers in cutaneous melanomas from Ras-Ink4a/Arf animals that were wild type (TM1-4) or heterozygous for Pten allele (TM5-9) relative to the ones from non-transformed INK4a-/- melanocytes.

C140 (Ras-Ink4a/Arf-Pten+/+) non-transformed melanocytes (A) and CN116 (Ras-Ink4a/Arf-Pten+/+) melanoma cell line (B-D) were infected with pSuper.Retro empty vector (EV) or two independent shRNAs targeting Pten- Sh4 and Sh11. CN44 (Pten+/-Ras-Ink4a/Arf-) melanoma cell line with Pten protein loss (E) was transduced with adenovirus expression GFP or PTEN. Western blots in A, B, and E show Pten level. Actin serves as a loading control).
(A, B &E) Boyden Chamber assay. Cell invasion through a Matrigel was quantified by counting cristal violet stained cells under a microscope (5 fields per well) (A), by measuring absorbance at O.D. 560nm after dye extraction (B), or by pixel quantification (E). Results reflect normalization for differences in proliferation (see Material and Methods). (C) Cell proliferation curve. The effect of Pten loss on growth of CN116 melanoma cells in 1% serum containing media was measured over 6 days by quantification of crystal violet staining. (D) Kaplan-Meier melanoma free survival analysis. SCID mice were subcutaneously injected into the flanks with half million cells of CN116 Ras-Ink4a/Arf melanoma cells infected with pSuper.Retro Sh4 (N=8), Sh11 (N=8) or empty vector (N=8). Inactivation of Pten by Sh11 resulted in earlier melanoma onset (P value=0.0081).

(A) Western blot analysis with indicated antibodies (top) and invasion chamber analysis (bottom) of NRAS mutated human melanoma cell WM1346 with siNT (non-targeting) control or with siRNA targeting PTEN (siPTEN).
(B) WM793A human melanoma cell lines harboring BRAF mutation and loss of PTEN expression were transduced with adenovirus encoding control GFP or with PTEN. Immunoblot (top left), Annexin V/PI apoptosis (bottom left), and invasion chamber analysis (right) are shown.