Supplementary figure legends:
Figure S1: (A) Cell morphology (shape) of fascin-overexpressed and vector control clone analysed by phase contrast microscopy. Scale bar:
100μm. (B) Representative image of the size and number of colonies formed in soft agar of the indicated clones. Scale bar: 100μm. (C) Western
blot analysis of fascin-overexpressed (AW-Fascin-1 and AW-Fascin-2) and vector control clones (AW-GFP-Cont) with antibodies to Ras, Rac,
Cdc42, RhoA and Vimentin. β-actin was used as a loading control.
Figure S2: Representative images of haematoxylin and eosin along with immunohistochemical staining with antibodies against fascin, K8 and
β4-integrin of paraffin embedded sections of human in oral tumor (A) and non malignant tissues (B).
Figure S3: Representative images of IHC staining with antibodies against fascin of paraffin embedded sections of primary tumor and lymph
node metastasized tumor of human OSCC tissues.
Figure S4: (A) Representative images of immunofluoroscence staining with antibodies against fascin, β4-integrin and K14 of paraffin embedded
sections of human oral tumor and fascin IF staining in non malignant tissues (B). Representative images of immunofluoroscence staining with
antibodies against β4-integrin and K1 of paraffin embedded sections of non malignant oral tissues.
Table S1. The Fascin overexpressed clones show an decrease in cell-cell adhesion. Cell adhesion was measured by the hanging drop assay as
described. 2 x 104 cells of the indicated knockdown clones were resuspended in 35μl of complete medium on the lid of a 24 well dish. 16 hours
later the cells were fixed and the number and area of aggregates in fifteen fields was measured. The numbers of aggregates of different sizes are
shown.
Construct
Aggregates per field
>105m2
105-2x104m2
<2x104-1x104m2
<104-3.5x103m2
AW-GFP-Cont
3
12
10
0
AW-Fascin-1
0
0
0
25
AW-Fascin-2
0
0
0
25
Table S2: Clinico-pathological parameters of the OSCC patients.
Clinico-pathological parameters
Age (Years)
Sex
Location
Thickness
Stages
Tumor
Size
Node Status
Differentiation
Bone
Perineural Invasion
<50
≥50
Male
Female
Tongue
BM
<2cm
≥2cm
I
II
III
IV
<T3
≥T3
NO
N1
N2
Poor+ Moderate
well
Positive
Negative
Yes
No
No of cases (131)
Percentage
75
56
102
29
61
70
92
39
11
17
18
85
48
83
47
34
50
124
7
33
69
28
92
57.25
42.75
77.87
22.14
46.56
53.44
70.23
29.77
8.40
12.98
13.74
64.89
36.65
63.35
35.88
25.95
38.17
94.65
5.34
25.19
52.67
21.37
70.23
Lympho vascular invasion
Perineural Extension
Cut Margin
Recurrence
Skin
Yes
No
Yes
No
Free
Closed
Invasion
Yes
No
Yes
No
3
112
55
62
118
8
5
49
78
11
68
2.29
85.50
41.98
47.33
90.08
6.11
3.82
38.58
61.42
8.40
51.91
Table S3: Correlations of fascin in combination with K8 and β4-integrin expression with clinico-pathological parameters of the OSCC
patients.
FASCIN AND β 4 INTEGRIN
Clinico-pathological
parameter
Age (Years)
Sex
Stages
Tumor
Size
Node Status
Differentiation
Perineural
Extension
Recurrence
<50
≥50
Male
Female
≥2cm
I/II
III/IV
T1/T2
T3/T4
NO
N1
N2
Poor+
Moderate
well
No
FASCIN AND K8
EXPRESSION
n
(123)
EXPRESSION
71
52
99
24
37
27
96
46
77
43
33
47
44
24
57
11
22
9
59
21
47
13
20
35
13
12
19
6
7
10
15
14
11
15
5
5
fascin(-)
β4 (+) or
fascin(-)
β4 (-)
14
16
23
7
8
8
22
11
19
15
8
7
116
66
24
26
fascin(+)
β4(+)
fascin(+)
β4 (-)
7
107
2
59
1
24
4
24
Yes
52
36
6
10
No
59
27
17
15
Yes
48
29
12
7
No
72
39
13
20
* Pearson Chi-Square; # Spearman Correlation (Ordinal by Ordinal)
P-value
0.202*
0.582*
0.032#
0.246#
0.001#
0.068#
0.028*
0.213*
n
(131)
75
56
102
29
39
28
103
48
83
47
34
50
48
22
58
14
23
9
63
21
51
17
21
34
16
10
19
7
8
10
16
14
12
13
5
8
fascin(-)
K8 (+) or
fascin(-)
K8 (-)
15
18
25
8
8
9
24
13
20
17
8
8
124
72
24
28
7
112
0
61
2
24
5
27
55
35
9
11
62
31
15
16
49
25
17
7
78
46
9
23
Fascin (+)
K8 (+)
fascin(+)
K8 (-)
P-value
0.285*
0.693*
0.020#
0.124#
0.002#
0.001#
0.323*
0.004*