Supplementary figure legends
Supplementary figure S1. UP-regulation of MMPs expression is independent of HPSE
extracellular activity
(A) RT-qPCR analysis of VEGF gene expression of melanoma cells upon siRNA
transfection. White bar: siCon transfected; black bar: siHPSE transfected. NS: no significant
difference; **, p < 0.01. (B-D) RT-qPCR analysis of MMP-9 (B), MMP-2 (C), and HPSE (D)
gene expression of human melanoma cells treated with unfractionated heparin. White bar:
non-treated; black bar: UFH treated. NS: no significant difference; **, p < 0.01. (E) Western
blot analysis of HPSE protein level of human melanoma cells treated with unfractionated
heparin. (F) Densitometry analysis of HPSE protein level as measured by western blot. White
bar: non-treated; black bar: UFH treated. NS: no significant difference.
Supplementary
figure
S2.
TNF-
induces
NF-B
activation
and
nuclear
translocalization in human melanoma cells
(A, B) Immunofluorescence stainings of HPSE (red) (A) or MMP-9 (green) (B) of siRNA
transfected human melanoma cells. Scale bar corresponds to 20 µm. (C) Immunofluorescence
stainings of NF-B p65 (red) of TNF- or non-treated human melanoma cells. Scale bar
corresponds to 20 µm. (D) Western blot analysis of NF-B p65 protein level in the cytosolic
and nuclear extracts of TNF- or non-treated human melanoma cells.
Supplementary figure S3. TNF- treatment promotes MMP-9 expression in human
melanoma cells
(A) Gelatin gel zymography analysis of MMP-9 activity of human melanoma cells upon
TNF- treatment. (B) Densitometry analysis of the MMP-9 zymography results. White bar:
treated with 1 ng/ml TNF-; grey bar: treated with 5 ng/ml TNF-; black bar: treated with 10
1
ng/ml TNF-. **, p < 0.01. (C) RT-qPCR analysis of MMP-9 gene expression of human
melanoma cells with TNF- treatment. White bar: non-treated; black bar: treated with 10
ng/ml TNF-. *, p < 0.05; **, p < 0.01. (D) Western blot analysis of HPSE protein level of
human melanoma cells treated with TNF- (10 ng/ml). (E) Densitometry analysis of HPSE
protein level as detected by western blot. White bar: non-treated; black bar: treated with TNF. NS: no significant difference; **, p < 0.01. (F) RT-qPCR analysis of HPSE gene
expression of TNF- treated human melanoma cells. White bar: non-treated; black bar:
treated with 10 ng/ml TNF-. NS: no significant difference; *, p < 0.05; **, p < 0.01.
Supplementary figure S4. Molecular interaction between albumin and DNA
(A) Two dimensional images obtained by atomic force microscopy of single albumin
molecules. (B) Two dimensional images obtained by atomic force microscopy of complexes
formed between DNA and albumin mixed at a molar ratio of 1.6 pmol albumin to 0.03 pmol
DNA. Scale bar corresponds to 50 nm.
Supplementary figure S5. Functional analysis of transfected mouse melanoma cells
(A) RT-qPCR analysis of HPSE gene expression of siRNA transfected B16F10 melanoma
cells. **, p < 0.01. (B) Analysis of the B16F10 melanoma cell proliferation after knockdown
of HPSE by WST-1 assay. Circle: siCon transfected; square: siHPSE transfected. (C) Mice
were intravenously (i.v.) injected with B16F10 cells (1.5 x 105) and euthanized after 14 days.
Metastatic foci were counted and macroscopic pictures of lungs were documented (n=5).
p=0.02.
Supplementary figure S6. Knockdown of HPSE inhibits ret xenograft growth as well as
angiogenesis but promotes lymph node metastasis
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(A) RT-qPCR analysis of MMP-9 gene expression of siRNA transfected ret melanoma cells.
**, p < 0.01. (B) Tumor volume of mice inoculated with transfected ret melanoma cells
(n=10). White circle: siCon transfected; black circle: siHPSE transfected. *, p < 0.05; **, p <
0.01. (C) Immunofluorescence stainings of CD31 (red) of ret tumor cryosections. Mosiac
images, composed of 48 single images, are taken from at least 10 fields of 5 different
xenografts. Scale bar corresponds to 500 µm. (D) Microvessel numbers counted in ret tumor
xenografts of at least 10 independent tumor sections of 5 different xenografts. **, p < 0.01.
(E) Mice were inoculated with siRNA transfected ret melanoma cells. Lymph nodes
(inguinal, axillary and mandibular) were collected and macroscopic pictures were taken (left
panel). Analysis of mice with lymph node metastasis or not was listed as bar graph (right
panel) (n=9). (F) Semi-quantitative analysis of protein expression in tumor tissue lysates from
transfected ret cell induced tumors. Lysates of 3 tumors of each group were pooled and
analyzed with a proteome profiler array. Differences of the protein amounts between the two
groups were displayed.
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