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 Panel 1 Panel 2 Panel 3 Panel 4
SKMEL-19
SKMEL-28
SKMEL-39
SKMEL-22
SKMEL-192
SKMEL-105
SKMEL-188
SKMEL-267
SKMEL-7
HCC 364
Colo205
A673
DU 4475
Panel 4
SKMEL-190
SKMEL-202
SKMEL-73
SKMEL-32
SKMEL-41
SKMEL-90
SKMEL-269
SKMEL-31
SKMEL-276
SKMEL-200
SKMEL-228
SKMEL-207
SKMEL-284
Panel 3
SKMEL-1
SKMEL-40
SKMEL-64
SKMEL-133
SKMEL-178
SKMEL-196
SKMEL-222
SKMEL-232
SKMEL-238
SKMEL-239
SKMEL-244
SKMEL-275
SKMEL-283
PTEN
pAkt (S473)
pAkt (T308)
β Actin
Panel 1
SKMEL-37
WM88
WM1361A
Malme3M
A2058
SKMEL-11
SKMEL-100
RKO-1
NPA
FRO
HT29
V600E BRAF Non melanoma
BRAF WT melanoma
V600E BRAF melanoma
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 B C SKMEL19/WTPTEN SKMEL207/723-724insTTPTEN
Copy number (log2)
Genome position (10q23.2-q23.31)
Copy number (log2)
Chromosome 10 position (mb)
SKMEL190
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 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. (V600EBRAF, PTEN null, RB1 wt)
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 PD (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 Malme3M
WM-88
A375
SKMEL-41
SKMEL-19
SKMEL-284
SKMEL-178
SKMEL-105
SKMEL-39
SKMEL-207
SKMEL-269
A2058
SKMEL-190
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 B MEL39/vector MEL39/E7 MEL39/E721-24
Supplemental Figure 6. Loss of RB1 function confers resistance to PLX 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 PLX4720 were 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 PD (25 mg/kg/day by oral gavage on days 1-5 each week) or vehicle only as control. Error bars represent standard error.