ADDITIONAL MATERIALS AND METHODS
In vitro culture of human lung biopsies
Small lung biopsy samples (1 to 5 mm3) were incubated in 200 µl PBS medium in the
upper side of a trans-well membrane with 0.4 µm pores (BD353495). Lower side of
the trans-well initially contained 100 µl PBS medium. After 5, 10, 30, and 60 minutes
incubation at room temperature, 20 µl culture mediums were collected in the lower
side of the membrane. Lung culture mediums were kept at -20°C until FADD
detection was performed. One hour-incubated lung samples were removed and kept
at -20°C until protein extraction was performed.
Western blot
Laemli buffer was added to 20 µl of collected supernatant culture or to 40 µg of cell
lystae, and boiled for 5 minutes. Then samples were subjected to SDS-PAGE and
transferred to PVDF membrane (NEN Life Sciences, Boston, Massachusetts). Each
gel run included a sample of A549 human carcinoma lung cells or of anti-Fas
antibody treated THP1 cells as positive control for FADD and caspase 8 western blot,
respectively. To insure standardization of the positive control, all A549/THP1 cell
proteins used were from a single, large protein preparation. After blotting,
membranes were probed with specific primary anti-FADD or anti-caspase 8
antibodies (Cell Signaling Technology, Ozyme, Saint-Quentin en Yvelines, France).
The proteins were visualized using the enhanced chemiluminescence technique
(Amersham Pharmacia Biotech, Orsay, France). Bands obtained were quantified by
densitometry using Fusion capt® and Bio1d® software (Vilber Lourmat, Marne la
Vallée, France).
Lactate dehydrogenase (LDH) assay
NSCLC were cultured 1 hour in PBS medium through a trans-well membrane with 0.4
µm pores. Culture medium was then collected in the bottom of the well. LDH activity
in the collected culture medium (secreted LDH) was determined by measuring the
change in absorbance at 490 nm using a LDH kit (Tox-7, Sigma-Aldrich) in a
microplate reader (EL8OO, Bio-Tek Instruments, Inc).
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SUPPLEMENTARY RESULTS
Presence of extracellular FADD in human NSCLC culture is not correlated with
tumor size
We examined whether the size of the tumor (T status) was correlated with the FADD
release process. By mean of Student’s t test and Spearman correlation test we did
not observe any significant differences between the level of FADD released by
differently sized T biopsies (P > 0.05 and 18%, respectively, Supplementary Figure 6
and not shown).
3
SUPPLEMENTARY FIGURES
Released FADD
(ng/mg proteins in the tissue)
30
25
20
15
10
5
0
T
NT
Released FADD
(AU/mg proteins in the tissue)
WESTERN BLOT
ELISA
250
200
150
100
50
0
T
NT
T
NT
T
NT
60
50
40
30
20
10
0
T
NT
Released FADD
(AU/mg proteins in the tissue)
Released FADD
(ng/mg proteins in the tissue)
Patient 30 : ADC stage II
140
120
100
80
60
40
20
0
100
80
60
40
20
0
T
NT
Released FADD
(AU/mg proteins in the tissue)
Released FADD
(ng/mg proteins in the tissue)
Patient 52 : ADC stage II
300
250
200
150
100
50
0
Patient 49 : ADC stage III
Supplementary Figure 1 Correlation between in vitro ADC biopsies' FADD release
determined by ELISA and Western blot analysis. FADD released by T (black) and by
distant NT (white) biopsies in vitro after one hour in vitro incubation was determined
by ELISA (left panel) and western blot (right panel). Histogram represents
quantification of bands obtained by western blot. Since lung biopsies’ size varied
among the patients, for each lung sample the released FADD protein quantity (in
arbitrary units) was reported to the total cytoplasm proteins quantity contained within
the sample (in mg), which reflected the biopsy size.
4
Released FADD
(ng/mg proteins in the tissue)
8
7
6
5
4
3
2
1
0
T
NT
Released FADD
(AU/mg proteins in the tissue)
WESTERN BLOT
ELISA
40
35
30
25
20
15
10
5
0
T
NT
7
6
5
4
3
2
1
0
T
NT
Released FADD
(AU/mg proteins in the tissue)
Released FADD
(ng/mg proteins in the tissue)
Patient 31 : SCC stage I
400
350
300
250
200
150
100
50
0
T
NT
T
NT
140
500
Released FADD
(AU/mg proteins in the
Released FADD
(ng/mg proteins in the tissue)
Patient 34 : SCC stage I
120
100
80
60
40
20
0
T
NT
400
300
200
100
0
Patient 68 : SCC stage III
Supplementary Figure 2 Correlation between in vitro SCC biopsies' FADD release
determined by ELISA and Western blot analysis. FADD released by T (black) and by
distant NT (white) biopsies in vitro after one hour in vitro incubation was determined
by ELISA (left panel) and western blot (right panel). Histogram represents
quantification of bands obtained by western blot. Since lung biopsies’ size varied
among the patients, for each lung sample the released FADD protein quantity (in
arbitrary units) was reported to the total cytoplasm proteins quantity contained within
the sample (in mg), which reflected the biopsy size.
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WESTERN BLOT
100
80
60
40
20
0
T1 T2 T3 T4 Mean T
Released FADD
(AU/mg proteins in the tissue)
Released FADD
(ng/mg proteins in the tissue)
ELISA
250
200
150
100
50
0
T1 T2 T3 T4
Mean T
Patient 50 : LCC stage II
Supplementary Figure 3 Correlation between in vitro LCC biopsies' FADD release
determined by ELISA and Western blot analysis. FADD released by T (black) and by
distant NT (white) biopsies in vitro after one hour in vitro incubation was determined
by ELISA (left panel) and western blot (right panel). Histogram represents
quantification of bands obtained by western blot. Since lung biopsies’ size varied
among the patients, for each lung sample the released FADD protein quantity (in
arbitrary units) was reported to the total cytoplasm proteins quantity contained within
the sample (in mg), which reflected the biopsy size.
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Released FADD
(AU/mg proteins in the tissue)
Released FADD
(AU/mg proteins in the tissue)
250
200
150
100
50
0
Incubation
time (min)
0.06
5 10 30 60
T
Released FADD
(AU/mg proteins in the tissue)
Patient 30 : ADC stage II
120
100
80
60
40
20
0
Incubation
time (min)
ND ND
5 10 30 60
5 10 30 60
T
NT
Patient 52 : ADC stage II
400
300
200
100
0
Incubation
time (min)
ND ND
5 10 30 60
5 10 30 60
T
NT
Released FADD
(AU/mg proteins in the tissue)
Released FADD
(AU/mg proteins in the tissue)
Patient 49 : ADC stage III
400
300
200
100
0
Incubation
time (min)
5 10 30 60
T
Patient 34 : SCC stage I
400
300
200
100
0
Incubation
time (min)
0.91
0.07
5 10 30 60
5 10 30 60
T
NT
Patient 68 : SCC stage III
Supplementary Figure 4 FADD protein release by human NSCLC is a rapid
process. Western blot analysis of FADD released by T (black) and by distant NT
(white) biopsies in vitro after different incubation times as indicated. Histogram
represents quantification of bands obtained by western blot. Since lung biopsies’ size
varied among the patients, for each lung sample the released FADD protein quantity
(in arbitrary units) was reported to the total cytoplasm proteins quantity contained
within the sample (in mg), which reflected the biopsy size. ND, not determined.
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Supplementary Figure 5 FADD protein release by human NSCLC is not a
consequence of cell death. Full length and p43/41 cleaved caspase 8 were detected
in one hour incubated NSCLC tumor biopsies. No p18 cleaved fragment could be
detected. In parallel, released LDH was measured in the culture medium of the one
hour-incubated NSCLC biopsies. ND, not determined; PT, proteins in the tissue.
8
T
1
2
3
4
n=
6
32
8
4
Supplementary Figure 6 Levels of released FADD were reanalyzed after the 50
NSCLC patients were classified according to their tumor size. Each diamond
represents a tumor patient biopsy. Bars represent the mean value. n represents the
number of patients.
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Supplementary Table 1 FADD protein release by human NSCLC is positively
correlated with the presence of vascular emboli
Supplementary Table 2 FADD protein release by human NSCLC is positively
correlated with the presence of vascular emboli
Supplementary Tables 1 and 2 FADD protein release by human NSCLC is
positively correlated with the presence of vascular emboli. For 26 patients of the
cohort, level of released FADD could be correlated with the presence (P) or absence
(A) of lymphatic (LVE), blood (BVE), intratumor, and peritumor vascular emboli. n
represents the number of patients. For other patients, histopathological report gave
no information on the presence or absence of emboli. In each case, P value indicates
the statistical difference between the amounts of FADD released by biopsies from
patients with or without emboli (P vs A). NS, not significant.
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