ba supplementary fig. s1. a, quantification of rip1 expression levels in melanocytic tumors. data...
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RIP
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me
an
IRS
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Compound Nevus
Dysplastic Nevus
Thin Primary
Thick Primary
Lymph Node
Metastases
Distant Metastases
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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. *P<0.05, 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. *P<0.05, Kruskal-Wallis test.
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Compound Nevus
Dysplastic Nevus
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Thick Primary
Lymph Node
Metastases
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RIP
1 p
ositi
ve c
ells
(%
)
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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.
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-MP
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l-RM
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- RIP1
- GAPDH
75kDa -
37kDa -
HE
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-DP
IgR
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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 (n=3, mean±S.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 (n=3, mean±S.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 (n=3, mean±S.E.M.). *P<0.05, Student’s t-test.
C
IgR3
Tetracyclinez-VAD-fmk
Nec-1
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MM200
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Tet.Ctrl
Tet.RIP1
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bili
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TetracyclineNec-1
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IgR3.RIP1MM200.RIP1
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Brd
UIn
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VectorRIP1-K45A
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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-κB reporter constructs for 24 hours were subjected to NF-κB reporter assays (n=3, mean±S.E.M.). *P<0.05, Student’s t-test.
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Melano. MM200 IgR3 Mel-RM ME4405
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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 (n=3, mean±S.E.M.).
VectorRIP1 cDNAz-VAD-fmk
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-+-
-++
Re
lativ
e c
ell
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bili
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%)
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A B
Mel
an
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Fresh Melanoma isolates
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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 (n=3, mean±S.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.
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Fresh Melanoma isolates
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lativ
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nd
ance
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RIP
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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 (n=3, mean±S.E.M.).
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IP1
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ActD
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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-κB reporter assays (n=3, mean±S.E.M.). *P<0.05, Student’s t-test. B, 72 hours after transfection, cells were subjected to BrdU incorporation assays (n=3, mean±S.E.M.). *P<0.05, Student’s t-test.
ME4405
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RIP1 cDNA
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lativ
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ctiv
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-κB
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RIP1 cDNA
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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 (<1mm
Breslow depth)20 13.7 81.3 (57-100)
Thick Primary (>1mm 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.
Supplementary Table S1, Summary of melanocytic tumors and their positivity for RIP1a