Supplementary Data
Loss of GDF10/BMP3b as a prognostic marker collaborates with TGFBR3 to enhance
chemotherapy resistance and epithelial-mesenchymal transition in oral squamous cell
carcinoma
Chieh-Wen Cheng#, Jenn-Ren Hsiao#, Chi-Chen Fan#, Yu-Kang Lo, Chi-Yuan Tzen, Li-Wha
Wu, Wei-Yu Fang, Ann-Joy Cheng, Chung-Hsing Chen, I-Shou Chang, Shih Sheng Jiang,
Jang-Yang Chang, and Alan Yueh-Luen Lee*
*: Corresponding author
Supplementary Materials and Methods
Cell culture
HSC-3, FADU, and KB cells were cultured in medium containing Dulbecco’s modified
Eagle’s essential medium (DMEM), supplemented with 5% fetal bovine serum (FBS) and 5%
super calf serum. The all medium contain penicillin 100 Units/mL, streptomycin 100 μg/mL
except mentioned. SCC-4, SCC-9, SCC-15, SCC-25, and SAS cells were cultured in medium
containing a 1:1 mixture of DMEM/F12 medium, supplemented with 10% FBS. OC3 cells
was cultured in medium containing a 1:1 mixture of DMEM/K-SFM medium, supplemented
with 10% FBS and OEC-M1 and YD-15 cells were cultured in medium containing RPMI
1640 medium, supplemented with 10% FBS. Oral pre-cancer lesion cell DOK (dysplastic oral
keratinocyte) was cultured in medium containing DMEM, supplemented with 10% FBS . Oral
keratinocyte normal cell lines, CGHNK2 and CGK6 , were grown in a culture medium
containing K-SFM medium, supplements for K-SFM, supplemented with G418 (400 μg/mL).
Whole cell lysate
The cell lysate preparation was performed as described previously [39]. Cells were lysed in
NETN (150 mM NaCl, 1 mM EDTA, 20 mM Tris-Cl pH 8.0, 0.5% NP-40 (v/v)) containing
protease and phosphatase inhibitors (50 mM sodium fluoride, NaF, 0.1 mM sodium
orthovanadate, NaVO4).
Western blot analysis
The cells were harvested by trypsinization and lysed with NETN buffer (20 mM Tris (pH 8.0),
1 mM EDTA, 150 mM NaCl, 0.5 % Nonidet P-40 (NP-40)) containing protease inhibitor
cocktail (Roche). The cell lysates were then centrifuged at 10,000 g at 4°C to obtain
solubilized cellular proteins. Protein was quantified with a bicinchoninic acid (BCA) protein
assay (Pierce, Rockford, IL) according to the manufacturer’s instructions. Proteins were
separated by 8% or 10% or 12% SDS-PAGE and electrotransfered to a polyvinylidene
fluoride membrane. Blots were probed with primary antibodies, followed by HRP-conjugated
goat anti-rabbit IgG (1: 5000, v/v) (Zymed, South San Francisco, CA, U.S.A.) or
HRP-conjugated goat anti-mouse IgG (1:5000, v/v) (Zymed, South San Francisco, CA,
U.S.A.). After washing with PBS containing 0.5% Tween-20, peroxidase activity was
assessed using enhanced chemiluminescence (PerkinElmer Life Science, Boston, MA,
U.S.A.). The intensities of the reaction bands were analyzed with the Image Gauge System
(Fuji, Tokyo, Japan).
Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA) and
reverse-transcribed at 37C with SuperScript II reverse transcriptase (Invitrogen). The
resulting cDNA was used as the template for PCR reactions. Real-time PCR (qPCR) reactions
were performed on a RotorGene 3000 system (Corbett Research, Mortlake, Australia) using
SYBR Green PCR Master Mix (Cambrex Co., East Rutherford, NJ). The sets of forward and
reverse primers, the corresponding PCR conditions, and the lengths of PCR products were
described as follows: GDF10 [5’-TGCTATGCAACAGATAAAGTTTGTAG-3’,
5’-TCCTGGTGTACCTGCCCTA-3’; 94C (30 sec), 60C (60 sec) and 72C (30 sec) for 40
cycles, 72 bp]; glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
[5’-GAAGGTGAAGGTCGGAGT-3’, 5’-GAAGAT GGTGATGGGATTTC-3’; 94C (30 sec),
52C (30 sec) and 72C (1 min) for 28 cycles, 220 bp]. All the PCR reactions were started at
94C for 5 min and terminated at 72C for 5 min. Finally, the data were analyzed using
RotorGene software v5.0 (Corbett Research). Differential RNA expressions between various
samples were calculated using GAPDH as an internal control.
Table S1. Clinicopathological characteristics in OSCC patients (n=122)
Variables
Number of patients (%)
Age (years)
 62
 62
Gender
Female
Male
Alcohol drinking
No
Yes
Betel quid chewing
No
Yes
Cigarette smoking
No
Yes
Differentiation
Well / Moderate
Poor
Tumor Size
 2 cm
 2 cm
Lymph node metastasis
No
Yes
TNM stage*
In situ
I
II
III
IV
Treatment
None
RT
CT
CT+RT
68 (55.7)
54 (44.3)
7 (5.7)
115 (94.3)
38 (31.1)
84 (68.9)
90 (73.8)
32 (26.2)
55 (45.1)
67 (54.9)
109 (89.3)
13 (10.7)
69 (56.6)
53 (43.4)
104 (85.2)
18 (14.8)
3 (2.5)
13 (10.8)
8 (6.7)
10 (8.3)
86 (71.7)
6
25
48
43
(4.9)
(20.5)
(39.3)
(35.2)
Recurrence
No
Yes
Death
No
Yes
80 (65.6)
42 (34.4)
60 (49.2)
62 (50.8)
*There are two patients who have no information of stage were excluded.
Table S2. Association of GDF10 expression with clinicopathological tumor
characteristics in OSCC patients (n=122)
Variables
Gdf10
High
Low
P-value
7
8
61
46
0.633
1
6
1
14
101
6
9
32
75
0.622
13
2
77
30
0.369
7
8
48
59
1
12
3
97
10
0.420
13
2
56
51
0.025
14
1
90
17
0.579
21
84
1
 II
3
12
Treatment
None
RT
CT
CT+RT
2
1
6
6
4
24
42
37
0.244
Age (years old)
 62
 62
Gender
Female
Male
Alcohol drinking
No
Yes
Betel quid chewing
No
Yes
Cigarette smoking
No
Yes
Differentiation
Well / Moderate
Poor
Tumor Size
 2 cm
 2 cm
Lymph node metastasis
No
Yes
TNM stage*
 II
Recurrence
No
Yes
Death
No
Yes
Patient number
13
2
67
40
0.122
7
8
53
54
1
15
107
Table S3. Association of GDF10 expression with clinicopathological tumor
characteristics in OSCC patients (n=62)
Variables
Gdf10
High
Low
P-value
4
4
27
27
1
0
3
1
8
51
3
5
15
39
0.882
7
1
36
18
0.434
2
6
21
33
0.714
6
2
47
7
0.716
7
1
23
31
0.046
8
0
41
13
0.273
1
7
8
46
1
8
54
Age (years old)
 62
 62
Gender
Female
Male
Alcohol drinking
No
Yes
Betel quid chewing
No
Yes
Cigarette smoking
No
Yes
Differentiation
Well / Moderate
Poor
Tumor Size
 2 cm
 2 cm
Lymph node metastasis
No
Yes
TNM stage*
 II
 II
Patient number
Supplementary Figure 1. Immunohistochemical analysis of GDF10 expression in OSCC
patients.
Representative immunohistochemical staining of GDF10 was performed by using
paraffin-embedded sections of OSCC and benign epithelium. The representative intensity of
immunostaining was classified as four levels: negative staining intensity (0) and positive
staining intensity, including weak (1 +), moderate (2 +), and strong (3 +) staining.
Microscopic magnification, ×400.
Supplementary Figure 2. Level of GDF10 afects cell growth, cell cycle progression and
the sensitivity to DNA-damaging agent
(A) Overexpression of GDF10 induces cell cycle arrest and decreases cell growth. 2 x 105
of OEC-M1 and OEC-M1/GDF10 cells were seeded. After 24, 48 and 72 hours, cell number
was analyzed by counting. **, p <0.01; ***, p <0.001.
(B) Knockdown of GDF10 increase the CPT sensitivity of SCC-9 cell line.
SCC-9 or shGD10 SCC-9cells were cultured in 96-well plates (1 × 104 cells/well) in triplicate
and then followed by treatment of increasing amount of CPT. After 1 day incubation, cell
viability was determined as described in Materials and Methods.
Supplementary Figure 3. The expression pattern of GDF10/BMP3b and TGFBR3 in
oral cancer cell lines.
The extracts of oral cancer cells were immunoblotted with an antibody to GDF10 and
TGFBR3, and antibody to GAPDH as a loading control.