B

1
B
A
Supplementary Fig. S1. A, quantification of RIP1
expression levels in melanocytic tumors. Data
shown are mean immunoreactive score (IRS)
S.E.M. of three individual experiments. Plt0.05,
Kruskal-Wallis test. B, quantification of RIP1
positive cells in melanocytic tumors. Data shown
are RIP1 positive cells () S.E.M. of three
individual experiments. Plt0.05, Kruskal-Wallis
test.
2
Supplementary Fig. S2. Melanocytes of different
origin express similarly lower levels of RIP1
than melanoma cell lines. Whole cell lysates
from HEMa-LP, HEMn-DP, HEMn-MP melanocytes and
IgR3, Mel-RM, ME4405 melanoma cells were
subjected to western blotting.
3
B
A
C
Supplementary Fig. S3. Increase in cell death
and proliferation triggered by overexpression of
RIP1 is independent of its kinase domain. A,
MM200.RIP1 and IgR3.RIP1 (Tet.RIP1), and their
corresponding vector control cells (Tet.Ctrl)
treated with tetracycline (1 µg/ml), z-VAD-fmk
(30 µM) or Necrostatin-1 (30 µM) as indicated for
24 hours were subjected to CellTiter-Glo cell
viability assays (n3, meanS.E.M.). B, MM200
and IgR3 cells transiently transfected with empty
vector or RIP1-K45A (kinase-dead RIP1) constructs
were subjected to CellTiter-Glo cell viability
assays (n3, meanS.E.M.). C, MM200.RIP1 and
IgR3.RIP1 treated with tetracycline (1 µg/ml) and
Necrostatin-1 (30 µM) as indicated for 72 hours
were subjected to BrdU incorporation assays (n3,
meanS.E.M.). Plt0.05, Students t-test.
4
Supplementary Fig. S4. NF-kB is constitutively
activated at high levels in melanoma cells
compared to melanocytes. HEMn-MP melanocytes,
MM200, IgR3, Mel-RM and ME4405 cells transiently
transfected with NF-?B reporter constructs for 24
hours were subjected to NF-?B reporter assays
(n3, meanS.E.M.). Plt0.05, Students t-test.
5
Supplementary Fig. S5. RIP1 overexpression
reduced viability of melanocytes by 10 in a
caspase-dependent manner. HEMn-MP melanocytes
transduced with empty vector or RIP1 cDNA
constructs were treated with z-VAD-fmk at 6 hours
after transduction. After another 18 hours, cells
were subjected to CellTiter-Glo cell viability
assays (n3, meanS.E.M.).
6
B
A
Supplementary Fig. S6. mRNA expression levels
and copy number variation of RIP1 in fresh
melanoma isolates. A, quantitative reverse
transcription-PCR analysis of total RNA extracted
from melanocytes and fresh melanoma isolates.
The relative abundance of RIP1 mRNA expression in
melanocytes was arbitrarily designated as 1 (n3,
meanS.E.M.). B, quantitative PCR analysis of
genomic DNA extracted from melanocytes and fresh
melanoma isolates. The relative copy number of
RIP1 in melanocytes was arbitrarily designated as
1.
7
Supplementary Fig. S7. The turnover rates of the
RIP1 mRNA remain comparable between melanoma and
melanocytes. Quantitative reverse
transcription-PCR analysis of total RNA extracted
from melanocytes, MM200 and Mel-RM cells treated
with 5 µg/ml actinomycin D (Act D) for indicated
periods. The relative abundance of RIP1 mRNA
expression in melanocytes at 0h was arbitrarily
designated as 1 (n3, meanS.E.M.).
8
A
B
Supplementary Figure S8. Knockdown of TNFR1
reduces activation of NF-kB and decreases
proliferation in melanoma cells, which can be
abolished by overexpression of RIP1. Mel-RM and
ME4405 cells were transiently transfected with
control siRNA (Ctrl siRNA), TNFR1 siRNA and RIP1
cDNA constructs as indicated. A, 24 hours after
transfection, cells were subjected to NF-?B
reporter assays (n3, meanS.E.M.). Plt0.05,
Students t-test. B, 72 hours after
transfection, cells were subjected to BrdU
incorporation assays (n3, meanS.E.M.).
Plt0.05, Students t-test.
9
Supplementary Table S1, Summary of melanocytic
tumors and their positivity for RIP1a
Melanocytic tumors Number RIP1 expression levels (IRS)b RIP1 positive melanoma cells
Compound Nevus 10 3.8 51c(37-68)d
Dysplastic Nevus 10 4.1 62.8 (40-78)
Thin Primary (lt1mm Breslow depth) 20 13.7 81.3 (57-100)
Thick Primary (gt1mm Breslow depth) 20 14.4 89.4 (65-100)
Lymph Node Metastases 20 12.8 85.8 (53-100)
Distant Metastases 20 11.4 75.9 (53-100)
a The cohort of the melanocytic tumors used in
this study was the same to that reported before
(27). b IRS Immunoreactive score. c Numbers
stand for the mean percentage of positive
cells. d Numbers in brackets represent the range
of positive cells.