Model of resistance to RAF inhibitors caused by splicing variants of BRAFV600E that lack the RASbinding domain a b Supplementary Fig 2 Effects of vemurafenib on ERK pathway activation and survival in Parental SKMEL239 cells and the ID: 816777
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Slide1
Supplementary Fig. 1
. Model of resistance to RAF inhibitors caused by splicing variants of BRAF(V600E) that lack the RAS-binding domain.
a
b
Slide2Supplementary Fig. 2.
Effects of vemurafenib on ERK pathway activation and survival in Parental SKMEL-239 cells and the vemurafenib-resistant clones. b
Vemurafenib
(2μM)
DMSO
P
C1
C2
C3
C4
C5
a
Slide3a
bSupplementary Figure 3. Vemurafenib-resistant cells remain sensitive to MEK inhibition.
Slide4Supplementary Fig.
4. Vemurafenib-resistant SKMEL-239 cells retain and express the BRAF(V600E) mutation.
SKEML-239 C3
T
A
T
SKMEL-239 C5
A
SKMEL-239 Parental
T
A
T
A
SKMEL-239 Parental
SKMEL-239 C5
SKMEL-239 C3
T
A
T
A
b
a
Genomic DNA
cDNA
Slide55
Supplementary Fig. 5. Evaluation of RTK activation and COT expression in the vemurafenib-resistant cells.
Par
C3
C5
EGFR
Eph B2
Eph B6
Insulin R
IGF-1R
ROR 1
Tie 1
PDGFR
β
a
b
SKMEL-22
SKMEL-28
SKMEL-188
SKMEL-222
SKMEL-275
SKMEL-239-Parental
239-C1
239-C2
239-C3
239-C4
239-C5
HT-29
Slide6NRAS
mtBRAF wtNRAS wt
BRAF
mutant (All V600E)
Supplementary Fig. 6.
p61BRAF(V600E) is not detected by western blot in a panel of
vemurafenib
treatment-naïve
melanoma cell lines.
SKMEL-239-Parental
239-C3
239-C5
267
Malme-3M
A375
SKMEL-188
SKMEL-11
SKMEL-28
SKMEL-19
SKMEL-41
SKMEL-39
SKMEL-239-Parental
239-C3
239-C5
SKMEL-30
SKMEL-103
SKMEL-187
SKMEL-36
WM1382
BRAF
β
-actin
SKMEL-22
SKMEL-1
SKMEL-40
SKMEL-100
SKMEL-222
SKMEL-275
SKMEL-188
SKMEL-64
Slide7Supplementary Fig.
7. Detected splicing variants encode for BRAF proteins with in-frame deletions.
Slide8Supplementary Fig. 8
. Quantitation of the transcript encoding p61BRAF(V600E) in the parental and vemurafenib-resistant cells.
Slide9HT-29 Colon BRAF(V600E)
Supplementary Fig. 9. Ectopic expression of p61BRAF(V600E) promotes resistance to vemurafenib.
a
SKMEL239- Parental
b
pMEK
pERK
pMEK
pERK
MEK
V5
BRAF
-
+
-
+
vemu
2
μ
M/1 hour
Short exp
Longexp
p61VE
Control
-
+
-
+
vemu
2
μ
M/1 hour
p61VE
Control
pMEK
pERK
MEK
V5
BRAF
Slide10p<0.05
p<0.05
a
b
Supplementary Fig.
10
.
ERK signaling and cell growth are
dependent on p61BRAF(V600E) in C3 cells.
c
d
C3 cells
C3 cells
Parental cells
C3 cells
Non targeting
CRAF
ex[4-8]-2
vemu
2
μ
M/1 hour
-
+
-
+
-
+
pMEK
pERK
BRAF
CRAF
MEK
Non
targeting
ARAF
CRAF
JC-1
JC-2
ex[4-8]-1
ex[4-8]-2
JC 1+2
Ex[4-8]–1+2
pMEK
pERK
ARAF
BRAF
CRAF
Cyclin D1
p27
MEK
Non
targeting
CRAF
JC-2
ex[4-8]-1
Ex[4-8]-2
ARAF
pMEK
pERK
ARAF
BRAF
CRAF
Cyclin D1
p27
MEK
Slide11pMEK
V5
MEK
0 40 200 800
0 40 200 800
IP: BRAF(V600E)
IP:
p61
BRAF(V600E)
nM
vemurafenib
Supplementary Fig. 11.
In vitro
BRAF
kinase
assay in the presence of RAF inhibitor.
Slide12Supplementary Fig. 12.
BRAF(V600E) can activate ERK signaling as a monomerBRAF -/-
pMEK
pERK
V5
BRAF
MEK
Control
VE
VE/R509H
pMEK
pERK
V5
CRAF
MEK
A
RAF
Control
VE
VE/R509H
A
RAF-/-
CRAF-/-
MEFs
+/+
a
b
Slide13IP:FLAG -
Blot: V5IP:FLAG - Blot: FLAGIP:FLAG - Blot
: V5 (long exp.)
WCL Blot: FLAG
WCL Blot: V5
WCL
Blot: MEK
Control
p61VE
p61VE/R509H
p61VE/R509H
X2
p61VE/R509H
X3
Supplementary Fig. 13.
The R509H mutation
impairs dimerization
of p61BRAF(V600E).