Bizzozero et al., p 1
Supplementary Information
Acid sphingomyelinase determines melanoma progression and metastatic behaviour via the
microphtalmia-associated transcription factor signalling pathway.
Laura Bizzozero, Denise Cazzato, Davide Cervia, Emma Assi, Fabio Simbari, Fabio Pagni,
Clara De Palma, Antonella Monno, Chiara Verdelli, Patrizia Rovere Querini, Vincenzo
Russo, Emilio Clementi and Cristiana Perrotta
Supplementary Figure legends
Supplementary Figure S1. A-SMase quantification methods in human melanoma tissue array.
(A) Schematic representation of the quantitative assessment of IHC staining intensity method using
the ImageJ colou deconvolution plug-in. (B) Quantitative assessment of A-SMase immunoreactivity
on human melanoma tissue samples using the AxioVision 4.6 software. Values are expressed as
mean ± SEM (benign nevi, n = 15; primary melanomas, n = 70; lymph node metastases, n = 30).
Error bars: SEM. *p < 0.05, **p < 0.01; ***p < 0.001.
Supplementary Figure S2. B16 melanoma clones characterization. Immunofluorescence images
of four representative B16 black (B16-B9 and B16-B11) and four representative B16 white (B16W6 and B16-W1) clones stained with the melanoma cells marker Mel-A Ab. All the images shown
are representative of one out of three reproducible experiments.
Bizzozero et al., p 2
Supplementary Figure S3. Selection of the B16-W6 clone expressing A-SMase siRNA. (A, B and
C) Validation of smpd1 siRNA sequence specifity. (A) smpd1 siRNA sequence selection. B16-W6
cells were transiently transfected with the scrambled siRNA or with three independent smpd1
siRNAs and A-SMase expression was evaluated by western blotting and flow cytometry analysis.
Values are expressed as Relative Fluorescence Intensity (RFI) ± SEM (n = 3), normalised on
untransfected B16-W6 (CTR). Image shown is representative of one out three reproducible
experiments. (B and C) Off-target effect analysis of smpd1 silencing. (B) qPCR of smpd1, ghr, hprt
and rpl32 (unrelated genes) with gapdh used as internal control of B16-W6 cells transfected with
the scrambled siRNA and with smpd1siRNA 1 and 2. C) qPCR of gapdh using as internal standard
rpl32, rplp0 and hprt of B16-W6 cells transfected with the scrambled siRNA and with
smpd1siRNA 1 and 2 to validate gapdh as control of gene expression. Values are expressed as mean
± SEM (n = 3). (D and E) Analysis of A-SMase expression levels and of proliferation (MTT assay)
of B16-W6 cells transfected with the with the pSilencer4.1-CMV neo Negative Control plasmid
compared to the parental cell clone. Values are expressed as fold change over control (B1-W6) (n =
3). (F) Analysis of B16-W6 cells stably transfected with smdp1 siRNA1. Thirty stably transfected
clones were analysed. A-SMase expression was assessed by flow cytometry. Values are expressed
as Relative Fluorescence Intensity (RFI) ± SEM (n = 3), normalised on B16-W6 (control). *p <
0.05, **p < 0.01; ***p < 0.001.
Supplementary Figure S4. A-SMase expression regulate tyrosinase expression and modified cell
ceramide pattern. (A) reverse transcription PCR of Tyr and GAPDH of B16-B9, B16-W6 and B16W6_pSIL10 clones. Images shown are representative of one out of four reproducible experiments.
(B) Ceramide levels in B16-B9, B16-W6 and B16-W6_pSIL10 clones measured by mass
spectrometry as explained in Supplementary Materials and Methods.
Bizzozero et al., p 3
Supplementary Figure S5. A-SMase down-regulation triggers proliferation of murine melanoma
cells in vitro. (A) Soft agar assay for measurement of clonogenic capacity of B16-B9, B16-W6,
B16-W6_pSIL10. Images are representative of three reproducible experiments. (B) MTT analysis
of B16-B9, B16-W6 and B16-W6_pSIL10 cell viability following 24, 48 and 72 h of culture in
normal condition (medium containing 10% FBS, left panel) or in serum deprivation (medium
containing 2% FBS, right panel). Values are expressed as fold increase over control (time 0 for
each clone) ± SEM (n = 6). (C) Cell cycle analysis of B16-B9, B16-W6 and B16-W6_pSIL10 cell
cultured for 6, 24, 48 and 72 h in medium containing 10% FBS or 2% FBS, stained with propidium
iodide and analysed by flow cytometry. Graphs represent the percentage of cells in the G2 phase of
cell cycle. Values are expressed as mean ± SEM (n = 5). Asterisks in B and C indicate statistical
significance vs. B16-W6 cells; **p < 0.01; ***p < 0.001.
Supplementary Figure S6. A-SMase expression affects the metastatic potential of melanoma
cells in vitro.(A and B) Evaluation of metastatic potential in vitro. (A) Migration and invasion
assays in B16-B9, B16-W6 and B16-W6_pSIL10 cells. Cells were plated onto a polycarbonate
membrane insert (migration) or onto a Matrigel-coated polycarbonate membrane insert (invasion)
using a 3T3 fibroblast-conditioned medium as source of chemoattractants. After 6 h of incubation,
the cells that had migrated onto the lower surface of the membrane were stained with crystal violet
and visually counted in 10 random fields. Images are representative of four different reproducible
experiments. (B) Transmigration assay. A monolayer of H5V endothelial cells was seeded onto the
polycarbonate membrane. B16-B9, B16-W6 and B16-W6_pSIL10 were loaded with CFSE vital dye
and let to migrate through the H5V monolayer for 6 h. The migrated cells were analysed by
fluorometry. Values are expressed as mean ± SEM (n = 4). (C) qPCR of Mel A and Pax-3 of B16B9, B16-W6 and B16-W6_pSIL10 cells. Values are expressed as mean ± SEM (n = 4). (D) Scatter
plots showing correlation of metastasis associated genes between B16-B9, B16-W6, B16-
Bizzozero et al., p 4
W6_pSIL10 and B16-F10. (E) Heat map representing gene expression changes in B16W6_pSIL10, B16-B9 and B16-F10 vs. B16-W6 cells. Asterisks in A and B indicate statistical
significance vs. B16-W6 cells; ***p < 0.001
Supplementary Figure S7. Comparison of B16-F10 cells vs B16-B9, B16-W6, B16-W6_pSIL10.
(A, B and C) A-SMase expression by qPCR (A) and activity (B), and melanin content (C) of B16F10, B16-B9, B16-W6 and B16-W6_pSIL10 as in Figure 2i, j and k. Values are expressed as mean
± SEM (n = 3). (D) Western blotting of Mitf, GAPDH, phospho-ERK (p-ERK) and ERK performed
on B16-F10, B16-B9, B16-W6 and B16-W6_pSIL10. Images are representative of three different
reproducible experiments; *p < 0.05, **p < 0.01; ***p < 0.001.
Supplementary Figure S8. Validation of mouse B16 cells transfection and of PD98059
efficiency. (A) Western blotting of A-SMase, Mitf, phospho-ERK (p-ERK), ERK and GAPDH
performed on B16-W6_pSIL10 cells untreated (CTR), and transfected with the scrambled siRNA or
with a specific Mitf siRNA (siMitf) and with an empty pEF1 vector (pEF1) or containing A-SMase
cDNA (A-SMase). Images shown are representative of one out three reproducible experiments. (B)
Western blotting of phospho-ERK (p-ERK), ERK performed on B16 cells untransfected or
transfected with a A-SMase cDNA (A-SMase) in the presence or absence of PD98059 (40 µM).
Images shown are representative of one out three reproducible experiments.
Supplementary Figure S9. Functional Validation of mouse B16 cells transfection. (A, B and C)
Analysis of proliferation (MTT assay) apoptosis (evaluation of Annexin V positive cells), and
migration (transwell migration assay) performed on B16-B9 and B16-W6-pSIL10 cells transfected
Bizzozero et al., p 5
with the scrambled siRNA of Mitf (scrMitf) or the empty pEF1 vector (pEF1). Values are expressed
as fold change over control (B16-B9 CTR and B16-W6-pSIL10 CTR) (n = 3).
Supplementary Tables
Supplementary table S1
Organ
Pathology diagnosis
TNM
1
skin, head and neck
primary melanoma
T4N1M0
2
skin, buttocks
primary melanoma
T4N1M0
3
skin, back
primary melanoma
T4N0M0
4
skin, sole
primary melanoma
T4N0M0
5
skin, shoulder
primary melanoma
T4N0M0
6
skin, foot
primary melanoma
T4N0M0
7
skin, thigh
primary melanoma
T4N0M0
8
skin, leg (sparse)
primary melanoma
T4N0M0
9
skin, sole
primary melanoma
T2N0M0
10 skin, shoulder
primary melanoma
T4N0M0
11 skin, leg
primary melanoma
T3N0M0
12 skin, sole
primary melanoma
T4N0M0
13 skin, shoulder
primary melanoma
T4N0M0
14 skin, thumb
primary melanoma
T4N0M0
15 skin, buttocks
primary melanoma
T4N0M0
16 skin, abdominal wall
primary melanoma
T4N1M0
17 skin, heel
primary melanoma
T3N0M0
18 skin, chest wall
primary melanoma
T4N0M0
19 skin, anus
primary melanoma
T4N0M0
Bizzozero et al., p 6
20 skin, sole
primary melanoma
T1N0M0
21 skin, chest wall
primary melanoma
T4N0M0
22 skin, heel
primary melanoma
T4N0M0
23 skin, toe
primary melanoma
T4N0M0
24 vulva
primary melanoma
T4N0M0
25 skin, buttocks
primary melanoma
T4N1M0
26 skin, back
primary melanoma
T3N0M0
27 skin, thigh
primary melanoma
T2N0M0
28 skin, foot
primary melanoma
T4N0M0
29 skin, thigh
primary melanoma
T4N0M0
30 skin, buttocks (sparse) primary melanoma
T4N0M0
31 skin, scalp
primary melanoma
T4N0M0
32 skin, thumbs
primary melanoma
T3N0M0
33 vulva
primary melanoma
T4N0M0
34 vulva
primary melanoma
T1N0M0
35 skin, cheeck
primary melanoma
T4N0M0
36 skin, arm
primary melanoma
T2N0M0
37 skin, sole
primary melanoma
T4N0M0
38 skin, leg
primary melanoma
T3N0M0
39 skin, malleolus
primary melanoma
T4N0M0
40 skin, heel
primary melanoma
T4N0M0
41 skin, foot
primary melanoma
T4N2M0
42 skin, buttocks
primary melanoma
T4N0M0
43 skin, sole
primary melanoma
T4N0M0
44 skin, scalp
primary melanoma
T4N0M0
45 skin, scalp
primary melanoma
T4N0M0
46 skin, toe
primary melanoma
Bizzozero et al., p 7
47 eyeball
primary melanoma
48 skin, sole
primary melanoma
49 skin, hand
primary melanoma
50 skin, abdomen
primary melanoma
51 soft tissue, axilla
primary melanoma
T4NXM1a
52 soft tissue, popliteal
primary melanoma
T4NXM1a
53 soft tissue, leg
primary melanoma
T4NXM1a
54 soft tissue, axilla
primary melanoma
T4NXM1a
55 nasal cavity
primary melanoma
TXN1M0
56 skin, forearm
primary melanoma
T4NXM0
57 skin, leg
primary melanoma
T4NXM0
58 skin, axilla
primary melanoma
T4N2MX
59 skin, axilla
primary melanoma
T4N0M0
60 rectum
primary melanoma
61 skin, back
primary melanoma
T4N1M1a
62 skin, axilla
primary melanoma
T4N1MX
63 eyeball
primary melanoma
64 eyeball
primary melanoma
65 rectum
primary melanoma
66 nasal cavity
primary melanoma
67 skin, forearm
primary melanoma
68 skin, thigh
primary melanoma
69 maxilla
primary melanoma
70 maxilla
primary melanoma
1
lymph node
lymph node metastasis
2
lymph node
lymph node metastasis
3
lymph node
lymph node metastasis
T3aN0M0
Bizzozero et al., p 8
4
lymph node
lymph node metastasis
5
lymph node
lymph node metastasis
6
lymph node
lymph node metastasis
7
lymph node
lymph node metastasis
8
lymph node
lymph node metastasis
9
lymph node
lymph node metastasis
10 lymph node
lymph node metastasis
11 lymph node
lymph node metastasis
12 lymph node
lymph node metastasis
13 lymph node
lymph node metastasis
14 lymph node
lymph node metastasis
15 lymph node
lymph node metastasis
16 lymph node
lymph node metastasis
17 lymph node
lymph node metastasis
18 Lung
lung metastasis
19 lymph node
lymph node metastasis
20 Lung
lung metastasis
21 lymph node
lymph node metastasis
22 lymph node
lymph node metastasis
23 lymph node
lymph node metastasis
24 lymph node
lymph node metastasis
25 lymph node
lymph node metastasis
26 lymph node
lymph node metastasis
27 lymph node
lymph node metastasis
28 lymph node
lymph node metastasis
29 lymph node
lymph node metastasis
Bizzozero et al., p 9
30 lymph node
lymph node metastasis
1
skin, shoulder
benign nevus
2
skin, abdominal wall
benign nevus
3
skin, chest wall
benign nevus
4
skin, waist
benign nevus
5
skin, shoulder
benign nevus
6
skin, frontal region
benign nevus
7
skin, face
benign nevus
8
skin, scalp
benign nevus
9
skin, back
benign nevus
10 skin, leg
benign nevus
11 skin, face
benign nevus
12 skin, buttocks
benign nevus
13 skin, abdominal wall
benign nevus
14 skin, arm
benign nevus
15 skin, face
benign nevus
Human melanoma Tissue Array table.
Supplementary Table S2.
N° colonies/field
Area (µm2)
B16-B9
86.9 ± 19.2
64.44 ± 16,9
B16-W6
59.50 ± 10.43
29.90 ± 16.61
B16-W6_pSIL10
106.40 ± 12.50
74.10 ± 22.89
Det-mel
89.00 ± 13.7
40.55 ± 4.20
Bizzozero et al., p 10
MRS3
63.50 ± 14.76
5.85 ± 1.85
Soft agar assay quantification. Cells (1 x 104) were plated in 0.3% agarose containing 10% FBS
over 0.6% agarose containing 10% FBS. After 14 days of incubation at 37°C in 5% CO2, colonies
from 10 random fields were counted and colony areas were measured with the software ImageJ (n =
3).
Supplementary table S3
Array
Position
Gene
accession
Number
Gene
name
Description
A01
NM_007462
Apc
Adenomatosis polyposis coli
A02
NM_134155
Brms1
Breast cancer metastasis-suppressor 1
A03
NM_013654
Ccl7
Chemokine (C-C motif) ligand 7
A04
NM_009851
Cd44
CD44 antigen
A05
NM_009864
Cdh1
Cadherin 1
A06
NM_009866
Cdh11
Cadherin 11
A07
NM_007666
Cdh6
Cadherin 6
A08
NM_007667
Cdh8
Cadherin 8
A09
NM_009877
Cdkn2a
Cyclin-dependent kinase inhibitor 2A
A10
NM_145979
Chd4
Chromodomain helicase DNA binding
protein 4
A11
NM_009932
Col4a2
Collagen, type IV, alpha 2
A12
NM_007778
Csf1
Colony stimulating factor 1 (macrophage)
B01
NM_013502
Ctbp1
C-terminal binding protein 1
B02
NM_009818
Ctnna1
Catenin (cadherin associated protein), alpha 1
B03
NM_007802
Ctsk
Cathepsin K
B04
NM_009984
Ctsl
Cathepsin L
B05
NM_021704
Cxcl12
Chemokine (C-X-C motif) ligand 12
B06
NM_009911
Cxcr4
Chemokine (C-X-C motif) receptor 4
Bizzozero et al., p 11
B07
NM_026603
Denr
Density-regulated protein
B08
NM_015779
Ela2
Elastase 2, neutrophil
B09
NM_010142
Ephb2
Eph receptor B2
B10
NM_008815
Etv4
Ets variant gene 4 (E1A enhancer binding
protein, E1AF)
B11
NM_007968
Ewsr1
Ewing sarcoma breakpoint region 1
B12
NM_001081286
Fat1
FAT tumor suppressor homolog 1
(Drosophila)
C01
NM_008011
Fgfr4
Fibroblast growth factor receptor 4
C02
NM_008029
Flt4
FMS-like tyrosine kinase 4
C03
NM_010233
Fn1
Fibronectin 1
C04
NM_008761
Fxyd5
FXYD domain-containing ion transport
regulator 5
C05
NM_053110
Gpnmb
Glycoprotein (transmembrane) nmb
C06
NM_053244
Kiss1r
KISS1 receptor
C07
NM_010427
Hgf
Hepatocyte growth factor
C08
NM_152803
Hpse
Heparanase
C09
NM_008284
Hras1
Harvey rat sarcoma virus oncogene 1
C10
NM_016865
Htatip2
HIV-1 tat interactive protein 2, homolog
(human)
C11
NM_010512
Igf1
Insulin-like growth factor 1
C12
NM_008360
Il18
Interleukin 18
D01
NM_008361
Il1b
Interleukin 1 beta
D02
NM_009909
Il8rb
Interleukin 8 receptor, beta
D03
NM_008398
Itga7
Integrin alpha 7
D04
NM_016780
Itgb3
Integrin beta 3
D05
NM_007656
Cd82
CD82 antigen
D06
NM_178260
Kiss1
KiSS-1 metastasis-suppressor
D07
NM_021284
Kras
V-Ki-ras2 Kirsten rat sarcoma viral oncogene
homolog
D08
NM_011029
Rpsa
Ribosomal protein SA
Bizzozero et al., p 12
D09
NM_008506
Mycl1
V-myc myelocytomatosis viral oncogene
homolog 1, lung carcinoma derived (avian)
D10
NM_023061
Mcam
Melanoma cell adhesion molecule
D11
NM_010786
Mdm2
Transformed mouse 3T3 cell double minute 2
D12
NM_008591
Met
Met proto-oncogene
E01
NM_019471
Mmp10
Matrix metallopeptidase 10
E02
NM_008606
Mmp11
Matrix metallopeptidase 11
E03
NM_008607
Mmp13
Matrix metallopeptidase 13
E04
NM_008610
Mmp2
Matrix metallopeptidase 2
E05
NM_010809
Mmp3
Matrix metallopeptidase 3
E06
NM_010810
Mmp7
Matrix metallopeptidase 7
E07
NM_013599
Mmp9
Matrix metallopeptidase 9
E08
NM_054081
Mta1
Metastasis associated 1
E09
NM_144800
Mtss1
Metastasis suppressor 1
E10
NM_010849
Myc
Myelocytomatosis oncogene
E11
NM_010898
Nf2
Neurofibromatosis 2
E12
NM_008704
Nme1
Non-metastatic cells 1, protein (NM23A)
expressed in
F01
NM_008705
Nme2
Non-metastatic cells 2, protein (NM23B)
expressed in
F02
NM_019731
Nme4
Non-metastatic cells 4, protein expressed in
F03
NM_015743
Nr4a3
Nuclear receptor subfamily 4, group A,
member 3
F04
NM_175116
P2ry5
Purinergic receptor P2Y, G-protein coupled,
5
F05
NM_011113
Plaur
Plasminogen activator, urokinase receptor
F06
NM_008891
Pnn
Pinin
F07
NM_008960
Pten
Phosphatase and tensin homolog
F08
NM_009029
Rb1
Retinoblastoma 1
F09
NM_146095
Rorb
RAR-related orphan receptor beta
F10
NM_023871
Set
SET translocation
Bizzozero et al., p 13
F11
NM_010754
Smad2
MAD homolog 2 (Drosophila)
F12
NM_008540
Smad4
MAD homolog 4 (Drosophila)
G01
NM_009271
Src
Rous sarcoma oncogene
G02
NM_009217
Sstr2
Somatostatin receptor 2
G03
NM_011518
Syk
Spleen tyrosine kinase
G04
NM_013836
Tcf20
Transcription factor 20
G05
NM_011577
Tgfb1
Transforming growth factor, beta 1
G06
NM_011594
Timp2
Tissue inhibitor of metalloproteinase 2
G07
NM_011595
Timp3
Tissue inhibitor of metalloproteinase 3
G08
NM_080639
Timp4
Tissue inhibitor of metalloproteinase 4
G09
NM_009425
Tnfsf10
Tumor necrosis factor (ligand) superfamily,
member 10
G10
NM_011640
Trp53
Transformation related protein 53
G11
NM_011648
Tshr
Thyroid stimulating hormone receptor
G12
NM_009505
Vegfa
Vascular endothelial growth factor A
H01
NM_010368
Gusb
Glucuronidase, beta
H02
NM_013556
Hprt1
Hypoxanthine guanine phosphoribosyl
transferase 1
H03
NM_008302
Hsp90ab1
Heat shock protein 90 alpha (cytosolic), class
B member 1
H04
NM_008084
Gapdh
Glyceraldehyde-3-phosphate dehydrogenase
H05
NM_007393
Actb
Actin, beta
Gene and accession number table of the mouse tumour metastasis array. H01-05: housekeeping genes.
Supplementary Materials and Methods
Materials
The following reagents were purchased as indicated: the Malignant melanoma, metastatic malignant
melanoma and benign nevus tissue array M1003 from US Biomax, Inc.; the Malignant melanoma
Bizzozero et al., p 14
Tissue array CK2 from Super Bio Chips; the polyclonal antibody (Ab) against A-SMase from Areta
international; the polyclonal Ab against Melan-A from Santa Cruz Biotechnology, the polyclonal
Ab against Ki67 and the monoclonal Ab against Mitf from Abcam; the polyclonal Ab against CD31
from BD Biosciences Pharmingen; biotinylated secondary Ab from Dako; the polyclonal Abs
against Bcl-2, CDK2, phospho-ERK, ERK and the anti-rabbit Ab HRP-conjugated from Cell
Signaling Technology; the anti-mouse Ab HRP-conjugated and Clarity Western Blotting ECL
substrate from Bio.Rad; Hematoxilin/Eosin and Masson-Fontana staining solutions from BioOptica.
Reagents for cell cultures were from Euroclone; Bicinchoninic Acid kit from Thermo Scientific;
Fluorescein isothiocianate (FITC)-labeled human recombinant Annexin V from Bender
MedSystem; Spmd1 siRNAs, Mitf siRNA, pSilencer4.1-CMV neo kit and the scrambled controls
siRNA, goat Alexa Fluor 546, Lipofectamine RNAiMAX Transfection Reagent, Cell trace
Carboxyfluorescein Succinimidyl ester (CFSE) proliferation kit, pEF1/Myc plasmid, Trizol
Reagents from Life Technologies; Fugene Transfection Reagent from Roche; RNeasy Micro kit
from Qiagen; ImProm-II™ Reverse Transcription System from Promega; Synthetic melanin,
MG132, PD98059and all other reagents from Sigma-Aldrich.
Immunohystochemistry and immunofluorescence quantification
Immunoreactivity was quantified with the NIH Image J software using the colour-deconvolution
plug-in that has a built-in vector for separating hematoxylin (H) and diaminobenzidine (DAB)
stainings. After colour deconvolution DAB images are processed separately. By using five random
test samples stained for every primary antibody, suitable threshold levels DAB were determined and
kept constant for all analysis. Thresholding creates binary DAB positive masks in which DABpositive pixels are pseudo-coloured with black, while the background is indicated with white colour
(Supplementary Figure S2A). The extent of staining is calculated as integrated optical density
(IOD), which is equal to the area × average density of image occupied by immunoreactivity (1) and
represented in graph as the mean ± SEM.
Bizzozero et al., p 15
To verify the reproducibility of the analysis, two blinded operators performed the same
measurements for Human Melanoma Tissue Arrays using also the computer-assisted imaging
analysis AxioVision Rel.4.6 (Carl Zeiss)(2). The analysis carried out with both methods includes
only the intact spots on the array (115 out of 159).
Immunofluorescence was quantified with MacBiophotonics Image J. The RGB images were splitted
to the respective red, green and blue image components. The red images were analysed using
thresholding as described for immunohistochemistry. The extent of staining is calculated as IOD
and represented in graph as the mean ± SEM.
Cell Culture and Treatments
Human melanoma cells Det-mel, GR4, Gian-mel, and MSR3 were established in the lab of Dr.
Vincenzo Russo (3-5). Murine melanoma cell lines B16-F10 and B16-F1 were obtained from
American Type Culture Collectionand were periodically cultured in our laboratory for the last 10
years. Cells were authenticated by isoenzymology and were routinely tested for Mycoplasma using
a MycoAlert mycoplasma detection kit (BioWhittaker-Lonza). Cells were cultured in Iscove’s
supplemented with 10% heat-inactivated foetal bovine serum (FBS), glutamine (200 mM),
penicillin/streptavidin (100 U/ml), 1% Hepes 1M pH 7.4 and grown at 37 °C in a humidified
atmosphere containing 5% CO2. The experiments for the evaluation of ERK phosphorylation and
Mitf degradation were carried out in the presence or absence of PD98059 (40 µM) for 1 h or
MG132 (1 µM) for 3 h, after 2 h of serum starvation.
Western blotting
Cells were homogenised in 50 mM Tris-HCl pH 7.4, 150 mMNaCl, 1% NP40, 0.25% Nadeoxycholate, and a protease inhibitor mixture and centrifuged at 1,500 × g for 5 min at 4 °C to
discard cellular debris. After separation by SDS-PAGE, polypeptides were electrophoretically
transferred to nitrocellulose filters (Whatman), and antigens were revealed by the respective
Bizzozero et al., p 16
primary Abs and the appropriate secondary HRP-conjugated goat anti-rabbit or anti-mouse Abs.
Proteins were visualised using ECL and exposure to autoradiography Cl-Xposure films (Thermo
Fisher Scientific) or with a Bio-Rad ChemiDoc MP imaging system (6).
Masson Fontana staining
The Fontana-Masson Stain is specific for melanin and "argentaffin granules". At pH 4, melanin
granules reduce silver nitrate to metallic silver, a histochemical reaction that reveals accumulation
of black material wherever melanin is located. Slices from all tumours obtained from pigmented
and not pigmented B16 clones were stained with Masson Fontana Kit following the standard
protocol(7).
Melanin content
1 x 105 cells were solubilized in 200 µl of 1N NaOH and 10% Dimethylsulfoxide (DMSO) for 2 h
at 80°C. Similarly, a standard curve using synthetic melanin covering the concentration range of 020 µg/ml was prepared. Sample and standard tubes were then centrifuged at 12,000 × g for 10 min
at RT, and supernatants transferred to a 96 multi-well plate. The absorbance of the supernatants was
measured at 410 nm and melanin content was determined using standard curve. Values were
expressed as µg per 1 x 105 cells (8).
Acid sphingomyelinase activity
Cells (2 × 106 cells/ml) were homogenised with 0.2% Triton-X100 in H2O, supplemented with N[methyl-14C]sphingomyelin (55 mCi/mmol; 50,000 dpm/assay; 0.3 mmol/assay), and A-SMase
activity was determined by measuring the conversion of sphingomyelin to phosphorylcholine
without added Zn2+ as previously described (9).
Bizzozero et al., p 17
LC-MS Analysis of Sphingolipids
Cells were pelleted, washed in PBS, and transferred to glass vials. Sphingolipid extracts, fortified
with internal standards were prepared and analysed as described (10). The LC-mass spectrometer
consisted of a Waters Aquity UPLC system connected to a Waters LCT Premier orthogonal
accelerated time-of-flight mass spectrometer (Waters), operated in positive electrospray ionisation
mode. Full scan spectra from 50 to 1,500 Da were acquired, and individual spectra were summed to
produce data points each 0.2 s. Mass accuracy and reproducibility were maintained by using an
independent reference spray by the LockSpray interference. The analytical column was a 100-mm
×2.1-mm inner diameter, 1.7-mm C8 Acquity UPLC BEH (Waters). The two mobile phases were:
phase A, methanol/water/formic acid (74/25/1 v/v/v); phase B, methanol/formic acid (99/1 v/v),
both containing also 5 mm ammonium formate. A linear gradient was programmed as follows: 0.0
min, 80 % B; 3 min, 90 % B; 6 min, 90 % B; 15 min, 99 % B; 18 min, 99 % B; 20 min, 80% B. The
flow rate was 0.3 ml/min. The column was held at 308 °C. Quantification was carried out using the
extracted ion chromatogram of each compound, using 50-mDa windows. The linear dynamic range
was determined by injecting standard mixtures. Positive identification of compounds was based on
the accurate mass measurement with an error < 5 ppm and its LC retention time compared with that
of a standard (< 2%).
Flow cytometry
A-SMase expression was analysed by flow cytometry using a Fluorescence-Activated Cell Sorter
(FACS) (FC500 Dual Laser system, Beckman Coulter) (6). To this purpose cells were trypsinized,
washed twice with PBS and fixed for 5 min at 4°C with 4% paraformaldehyde. Aspecific sites were
blocked incubating samples for 20 min at room temperature in 10% goat serum, 1% BSA, 0.1%
saponin in PBS. Cells were then incubated with the specific primary Ab against A-SMase in 1%
BSA, 0.1% saponin in PBS for 1 h at room temperature. For fluorescent detection appropriate
secondary Ab conjugated with Alexa 546 was used.
Bizzozero et al., p 18
Apoptosis detection
Apoptosis was analysed by flow cytometry as described previously(6) .Phosphatidylserine exposure
on the outer leaflet of the plasma membrane in PI-excluding cells was detected by the analysis of
cells stained for 15 min with FITC-labeled annexin V (1 μg/ml) and analysed by the FCS Express
software, version 3 (De Novo Software).
Proliferation assay
Proliferation rate was evaluated through the cell viability assay MTT and cell cycle analysis (6, 11).
For the MTT assay, cells (2 x 104) were plated in 96 well microplate and incubated for 16h in
medium without serum to synchronize cell cycle; cells were then cultured in complete growth
medium containing 10% FBS(normal condition) or in medium containing 2% FBS (serum
deprivation condition) for the indicated times. Elimination of serum from culture medium makes the
population of proliferating cells more homogenous, since they withdraw from the cell cycle to enter
the quiescent G0/G1 phase. Tumour cells undergoing serum starvation in vitro partially mimic
metabolically stressed cells trying to adjust to a changed environment in vivo by inducing signal
transduction and gene expression so that the tumour continues to grow. Indeed, serum starvation is
often used as an experimental model to recreate a poorly vascularised, nutrient-, growth factordeficient core of tumours, although it has been recently clarified that physiological extrapolations of
results obtained from serum-starved cells should be subject to constant scrutiny. Briefly, cells were
washed and fresh medium containing MTT (0.5 mg/ml) was added in each well. After 4h incubation
at 37 °C, the supernatant was gently removed and formazan crystals were dissolved in DMSO.
Absorbance was recorded at 570 nm with correction at 690 nm using a microplate reader (Glomax
Multi Detection System, Promega). For cell cycle analysis 1 x 106 cells were plated in a 6 well plate
and cultured as before, then harvested and fixed with 70 % ethanol for 2 h at -20°C. cells were
resuspended in PBS containing 200 µg/ml Rnase, 0.1% of NP40 and 20µg/ml PI for 30 min on ice
Bizzozero et al., p 19
and analysed for DNA content by quantifying the red fluorescence. The percentage of cells in the
G0/G1, S, or G2/M phases of cell cycle were determined by analysis of the results using the FlowJo
software version 7.5.5 (Tree Star).
Soft agar assay.
Soft agar assay was used to determine the efficiency of anchorage-independent growth. Cells (1 ×
104) were plated in 4 ml 0.3% agar in Iscove’s medium with 10 % FBS overlaid onto a solid layer
of 2 ml 0.6 % agar in Iscove’s supplemented with 10 % FBS. Cultures were maintained for 2 weeks
and the colonies obtained were stained with 0.005 % Crystal Violet (Sigma-Aldrich) and counted
using a dissecting microscope.
In vitro migration, invasion and transmigration assays
Cell migration was assayed in a 6-well plate in which transwell inserts with an 8-μm pore size
polycarbonate membrane (Corning) were placed. B16 cells (1 ×105) seeded into the upper
chambers, while 3T3 fibroblast-conditioned medium was placed in the lower compartment as a
source of chemoattractants. In the invasion assay the polycarbonate membrane of the upper
chambers was coated with Matrigel (BD Biosciences). Cells were allowed to migrate for 6 h and
those migrated in the lower side of the filter were fixed in 4 % paraformaldehyde and stained with
crystal violet [0.1% in methanol-water (2:8) solution]. The number of migrated cells was measured
using an inverted microscope and by counting 5-10 random fields at 20× magnification. In the
transmigration assay mouse microendothelial H5V cells were plated on the Matrigel-coated
transwell membrane (1× 105 cells/dish) and grown in DMEM containing 10 % FBS until they
reached confluence. Melanoma cells (1 × 105) loaded with CFSE were overlaid on the endothelial
monolayer and transmigration allowed to occur for 6 h. Cells on the lower side of the membrane
were collected and quantified by fluorometric determination (Ex: 490 nm, Em: 510–570 nm) using
the Glomax Multi Detection System (12).
Bizzozero et al., p 20
Reverse Transcription-PCR and Quantitative Real Time-PCR
Total RNA from cells and tissues was extracted with the High Pure RNA Isolation Kit and High
Pure RNA Tissue Kit (Roche), according to the manufacturer’s recommended procedure. After
solubilization in RNase-free water, total RNA was quantified by the Nanodrop 2000
spectrophotometer (Thermo Fisher Scientific). First-strand cDNA was generated from 1 μg of total
RNA using ImProm-II Reverse Transcription System (Promega). For Tyr levels evaluation RTPCR reactions were carried out using 1 μl of cDNA and the GoTaq Green Master Mix (Promega),
containing 500 nM of appropriate primers (Tyr: forward 5’-GGCCAGCTTTCAGGCAGAGGT-3’
and
reverse
5’-TGGTGCTTCATGGGCAAAATC-3’;
TGAAGGTCGGTGTGAACGGATTTG–3’
GAPDH:
and
reverse
forward5’–
5’–
CATGTAGGCCATGAGGTCCACCAC–3’) as follows: denaturation at 94 °C for 60s, 30 cycles of
annealing at 57°C for 30 s and elongation at 72° C for 60 s. Digital images of the bands were
analysed and quantified using NIH ImageJ software and each signal intensity was normalised to
GAPDH expression as a control. qPCR was performed using Light Cycler 480 SYBR Green I
Master (Roche) on Roche Light Cycler 480 Instrument, according to manufacturer’s recommended
procedure. As shown in Table below, a set of primer pairs were designed to hybridize to unique
regions of the appropriate gene sequence. All reactions were run as triplicates. The melt-curve
analysis was performed at the end of each experiment to verify that a single product per primer pair
was amplified. As to control experiments, gel electrophoresis was also performed to verify the
specificity and size of the amplified quantitative Real Time-PCR products. Samples were analysed
using the Roche Light Cycler 480 Software (release 1.5.0) and the second derivative maximum
method. The fold increase or decrease was determined relative to a control after normalising to
GAPDH (internal standard) through the use of the formula 2-ΔΔCT (13-14).
Bizzozero et al., p 21
Primer pairs designed for quantitative Real Time PCR analysis
Name
Symbol
Gene accession N° Primer sequence
NM_007475
36B4
rlpl0
NM_011421
A-SMase smpd1
c-met
met
NM_008591
GAPDH
gapdh
NM_008084
GH
receptor
ghr
NM_010284
HPRT
hprt
NM_013556
Melan-A mlana
NM_029993
NM_001113198
MITF
mitf
NM_008601
NM_001178049
Pax-3
pax3
NM_008781
RPL32
rpl32
NM_172086
Tyr
tyr
NM_011661
F: 5’-AGGATATGGGATTCGGTCTCTTC-3’
R: 5’-TCATCCTGCTTAAGTGAACAAACT-3’
F: 5’-TGGGACTCCTTTGGATGGG-3’
R: 5’-CGGCGCTATGGCACTGAAT-3’
F: 5’-CCCCAACTTCACGGCAGAAA-3’
R: 5’-GGCTCCGAGATAAATATGATGGC-3’
F: 5’-ACCCAGAAGACTGTGGATGG-3’
R: 5’-ACACATTGGGGGTAGGAACA-3’
F: 5’-ACAGTGCCTACTTTTGTGAGTC-3’
R:5’-GTAGTGGTAAGGCTTTCTGTGG-3’
F: 5’-TCAGTCAACGGGGGACATAAA-3’
R:5’-GGGGCTGTACTGCTTAACCAG-3’
F: 5’-AGACGCTCCTATGTCACTGCT-3’
R:5’-TCAAGGTTCTGTATCCACTTCGT-3’
F: 5’-CCAACAGCCCTATGGCTATGC-3’
R: 5’-CTGGGCACTCACTCTCTGC-3’
F: 5’-TTTCACCTCAGGTAATGGGACT-3’
R: 5’-GAACGTCCAAGGCTTACTTTGT-3’
F: 5’-TTAAGCGAAACTGGCGGAAAC-3’
R: 5’-TTGTTGCTCCCATAACCGATG-3’
F: 5’-GACATTGATTTTGCCCATGAAGCACC-3’
R: 5’-GATGCTGGGCTGAGTAAGTTAGG-3’
*
http://pga.mgh.harvard.edu/primerbank/
#
http://medgen.ugent.be/rtprimerdb/index.php
Amplicon Source
143bp
(15)
134 bp
PrimerBank*
70 bp
PrimerBank*
172 bp
RTPrimerDB#
133 bp
PrimerBank*
142 bp
PrimerBank*
121 bp
PrimerBank*
99 bp
PrimerBank*
275 bp
PrimerBank*
100bp
PrimerBank*
212 bp
RTPrimerDB#
Bizzozero et al., p 22
Cell transfection and RNA Interference
B16-B9, B16-W6_pSIL10 and Det-mel cells were transiently transfected with a pEF1/Myc plasmid
containing cDNA for A-SMase, using using Fugene transfection reagent according to the
manufacturer’s protocol. The Silencer® Select Pre-designed siRNA specific for Mitf (sense: 5’GGACAAUCACAACUUGAUUtt-3’/ antisense: 5’-AAUCAAGUUGUGAUUGUCCtt-3’) was
used to downregulate Mitf expression in murine melanoma cells. Briefly, B16-B9 and B16W6_pSIL10 cells seeded at 40% confluence were transfected when at 60 % confluence with the
siRNAs using Lipofectamine RNAiMAX tranfection reagent according to the manufacturer’s
protocol. B16-B9 and B16-W6-pSIL10 cells transfected with the empty pEF1/Myc vector or the
Silencer® Negative Control siRNA (scramble) were also generated. In these systems, no
modification of A-SMase and Mitf expression was observed (Supplementary Figure S8 and data not
shown). In addition, as shown in Supplementary Figure S9, B16- and B16-W6-pSIL10 cells
transfected with the empty pEF1/Myc vector or scrambled siRNA did not differ from the untreated
cells in terms of proliferative, apoptotic, and migratory capacity.
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