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SKMEL-19: T1799A (V600E), homozygous

Supplemental Figure 1. SKMEL-19: T1799A (V600E), homozygous. GTG -> GAG. SKMEL-32: T1799A (V600E), heterozygous. GTG -> GAG.

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SKMEL-19: T1799A (V600E), homozygous

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  1. Supplemental Figure 1 SKMEL-19: T1799A (V600E), homozygous GTG -> GAG SKMEL-32: T1799A (V600E), heterozygous GTG -> GAG Supplemental Figure 1. Representative Mass Spectrometry and Sanger traces for the SKMEL-19 and SKMEL-32 cell lines. SKMEL-19 harbored a homozygous mutation of BRAF (T1799A, V600E) whereas SKMEL-32 harbored a heterozygous mutation.

  2. Supplemental Figure 2 V600E BRAF melanoma BRAF WT melanoma SKMEL-100 SKMEL-37 SKMEL-11 SKMEL-39 WM1361A V600E BRAF Non melanoma Malme3M RKO-1 A2058 WM88 HT29 NPA FRO PTEN pAkt (S473) Panel 1 pAkt (T308) β Actin SKMEL-192 SKMEL-105 SKMEL-188 SKMEL-267 SKMEL-19 SKMEL-28 SKMEL-39 SKMEL-22 SKMEL-7 HCC 364 DU 4475 Colo205 A673 PTEN pAkt (S473) Panel 2 pAkt (T308) β Actin SKMEL-133 SKMEL-178 SKMEL-196 SKMEL-222 SKMEL-232 SKMEL-238 SKMEL-239 SKMEL-244 SKMEL-275 SKMEL-283 SKMEL-40 SKMEL-64 SKMEL-1 PTEN Panel 3 pAkt (S473) β Actin SKMEL-202 SKMEL-269 SKMEL-276 SKMEL-200 SKMEL-228 SKMEL-207 SKMEL-190 SKMEL-284 SKMEL-73 SKMEL-32 SKMEL-41 SKMEL-90 SKMEL-31 PTEN Panel 4 pAkt (S473) β Actin Supplemental Figure 2. PTEN expression in the class of V600EBRAF mutant cell lines. Forty-eight V600EBRAF mutant cell lines (40 melanoma and 8 non-melanoma) were assessed for PTEN and phosphorylated AKT expression. Nine PTEN null cell lines (also shown in Figure 1) were identified and used in subsequent studies. The SKMEL-39 cell line (V600EBRAF, PTEN null) is included in both panels 1 and 2. WM1361A and SKMEL-31 are BRAF wild-type.

  3. Supplemental Figure 3 A SKMEL19/WTPTEN T G SKMEL207/723-724insTTPTEN T T T G B C Copy number (log2) Copy number (log2) SKMEL190 Genome position (10q23.2-q23.31) Chromosome 10 position (mb) Supplemental Figure 3. Loss of PTEN expression in melanoma cell lines was the result of mutation or focal deletion.A. Sanger sequencing of cDNA generated from the SKMEL-207 identifying a homozygous two base pair insertion c.723_724insTT in the PTEN mRNA (shown) and corresponding exon sequencing data (not shown). This insertion results in early truncation of the protein. SKMEL-19 expresses wild type PTEN and is shown for comparison. B. Probe level and segmentation data for the SKMEL-133 and SKMEL-39 cell lines showing a focal deletion of the PTEN gene in SKMEL-133. SKMEL-39 cells are copy number neutral at the PTEN locus. C. Probe-level and segmentation data for the SKMEL-190 cell line from both Agilent 244K- and 1M-feature arrays (gray and blue/red respectively).

  4. Supplemental Figure 4 SKMEL-133 (V600EBRAF, PTEN null, RB1 wt) SKMEL-207 (V600EBRAF, PTEN/RB1 null) Supplemental Figure 4. Combined targeting of MEK and PI3K in V600EBRAF, PTEN-null melanoma cell lines. SKMEL-133 (V600EBRAF, PTEN null, RB1 wt) cells and SKMEL-207 (V600E BRAF, PTEN null, RB1 null) cells were treated with either the MEK inhibitor PD0325901 (50 nM), the PI3 kinase inhibitor PI-103 (2.5 µM) or the combination. Induction of cell death was determined by FACS following 6, 24 and 48 hours of drug exposure. Error bars represent standard deviation of triplicate experiments.

  5. Supplemental Figure 5 PTEN expressing PTEN null SKMEL-267 SKMEL-188 SKMEL-276 SKMEL-228 SKMEL-239 Malme3M SKMEL-32 SKMEL-11 SKMEL-28 SKMEL-1 WM-88 A375 SKMEL-284 SKMEL-39 A2058 SKMEL-41 SKMEL-178 SKMEL-207 SKMEL-190 SKMEL-19 SKMEL-105 SKMEL-269 SKMEL-133 PTEN pAkt(T308) pAkt(S473) Akt p16 p21 p27 RB1 p130 β Actin Supplemental Figure 5. RB1 and p16 loss are non-overlapping in melanoma cells lines harboring V600EBRAF mutations. A. Immunoblots of PTEN, pAkt, Akt, p16, p21, p27, RB1 and p130 in 23 melanoma cells lines harboring V600EBRAF mutations. Two cell lines, SKMEL-207 and A2058, expressed no RB1 protein. Both of these cell lines expressed wild-type p16.

  6. Supplemental Figure 6 A MEL39/vector MEL39/E7 MEL39/E721-24 B Supplemental Figure 6.Loss of RB1 function confers resistance to PLX4720. A. SKMEL-39 cells (V600EBRAF, PTEN-null, RB1 wt) were stably infected with retroviral E7, a dysfunctional E7∆21-24 mutant or empty vector. IC50 values for the RAF inhibitor PLX4720were determined by Alamar Blue assay on Day 5 of drug treatment. B. S Phase changes of the same cells as in A after 2.5 μM PLX4720 treatment for 48 hours. The error bars represent the standard deviation of three experiments.

  7. Supplemental Figure 7 SKMEL39 (V600EBRAF, PTEN null, RB1 wt) SKMEL133 (V600EBRAF, PTEN null, RB1 wt) Supplemental Figure 7. Effect of MEK inhibition on the growth of V600EBRAF/PTEN-null xenograft models. Established SKMEL-39 (A) and SKMEL-133 (B)xenografts were treated with PD0325901 (25 mg/kg/day by oral gavage on days 1-5 each week) or vehicle only as control. Error bars represent standard error.

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