Chen et al. 10.1073/pnas.0500397102. |
Supporting Methods
Supporting Table 1
Supporting Table 2
Supporting Figure 6
Supporting Table 3
Supporting Figure 6
Fig. 6.
FADD gene is not amplified in lung tumors showing a high level of expression, as compared with normal tissues by quantitative genomic PCR using GAPDH as the internal control. T, tumor; N, normal.
Table 1. Relationship between FADD and clinical–pathological characteristics of lung adenocarcinomas
Variables | n | P (protein) | P (mRNA) |
Age, yr | |||
<65 | 49 |
|
|
>65 | 44 | 0.110 | 0.180 |
Gender | |||
Female | 53 |
|
|
Male | 40 | 0.766 | 0.024 up |
Smoking status | |||
Smoking | 79 |
|
|
Nonsmoking | 10 | 0.807 | 0.132 |
Tumor vs. Normal | |||
Normal | 10 |
|
|
Tumor | 93 | 0.006 up | 0.0001 up |
Stage | |||
Stage I | 64 |
|
|
Stage III | 29 | 0.031 up | 0.001 up |
N status | |||
N0 | 68 |
|
|
N1–2 | 25 | 0.018 up | 0.004 up |
Classification | |||
Bronchioloalveolar | 14 |
|
|
Bronchial-derived | 76 | 0.185 | 0.001 up |
Differentiation | |||
Poor | 23 |
|
|
Well + Moderate | 70 | 0.520 | 0.050 |
Angiolymphatic invasion | |||
Yes | 16 | 0.038 up | 0.474 |
No | 77 |
|
|
Lymphocytic response | |||
Yes | 41 |
|
|
No | 52 | 0.011 up | 0.677 |
P53 nuclear accumulation | |||
Positive | 19 |
|
|
Negative | 72 | 0.507 | 0.865 |
K-ras 12th/13th codon mutation | |||
Positive | 36 | 0.904 | 0.008 up |
Negative | 41 |
|
|
Bold indicates P < 0.05 from F test (for native FADD protein) or t test (for FADD mRNA). Up indicates increase in expression in respective group.
Table 2. Primer sequence used for PCR amplification and sequencing of FADD
Primer | Direction | Primer sequence | Oligonucleotide length | Tm | Product size |
Amplification primers | |||||
FADD | Forward | 5'-ATGCGCGGGTCCCTTAGTTTTC-3' | 22 mer | 60.1 | 171 bp |
Reverse | 5'-CTCCGTGCGCCCTCTGTCC-3' | 19 mer | 60.1 |
| |
GAPDH | Forward | 5'-TCTTCCCACCCGCCCCAGTC-3' | 20 mer | 62.7 | 296 bp |
Reverse | 5'-TGCCCAACACCCCCAGTCATAC-3' | 22 mer | 60.3 |
| |
Sequencing primers | |||||
Exon 1 | Forward | 5'-ATGCGCGGGTCCCTTAGTTTTG-3' | 22 mer | 60.1 | 786 bp |
Reverse | 5'-CCCTCCCCCACACCTTCTCTG-3' | 21 mer | 59.3 |
| |
Exon 2 | Forward | 5'-CACTTGGCGTCTGTGCTGAAAAG-3' | 23 mer | 58.6 | 866 bp |
Reverse | 5'-ATGGGCTCTGGTCAAGGATGG-3' | 21 mer | 57.5 |
| |
Tm, melting temperature.
Table 3. Correlation between expression of FADD and cyclin mRNA in lung adenocarcinomas
Gene | r | P |
cyclin B1 | 0.53 | <0.01 |
cyclin D1 | 0.33 | <0.01 |
cyclin F | 0.21 |
|
cyclin A2 | 0.16 |
|
cyclin C | 0.14 |
|
cyclin E1 | 0.14 |
|
cyclin G1 | 0.08 |
|
cyclin H | 0.08 |
|
cyclin D3 | 0.07 |
|
cyclin I | –0.08 |
|
cyclin A1 | –0.11 |
|
cyclin D2 | –0.38 |
|
r
, Spearman’s correlation coefficient based on 86 tumor samples.Supporting Methods
Amplification and Mutation Analysis of FADD.
Primers used for amplification and mutation (sequence) analysis for Fas-associated death domain (FADD) are shown in Table 2. Lung adenocarcinoma and normal samples (24 pairs) were used for FADD amplification analysis by quantitative genomic-PCR using GAPDH as the internal control (1). Exons 1 and 2 of FADD were sequenced in the University of Michigan DNA Sequencing Core on Applied Biosystems model 3700 sequencers, by using protocols as specified by the manufacturer. Samples were cycle-sequenced with Applied Biosystems BigDye (version 1) dye-labeled terminators, and the resultant fragments were analyzed by capillary electrophoresis on an Applied Biosystems model 3700 genetic analyzer.Cell Culture, Transfection, and Reagents.
FADD-deficient and WT Jurkat A3 cells were from American Type Culture Collection). Cells were grown in RPMI medium 1640 containing 10% FBS, 100 units/ml penicillin, 100 mg/ml streptomycin sulfate, 1 mM sodium pyruvate, 10 mM Hepes, 292 mg/ml l-glutamine (Invitrogen) and maintained in a humidified incubator at 37°C and 5% CO2. Cells were transfected by using 16 ml of FuGene (Roche Diagnostics) and 4 mg of DNA mixed in 500 ml of optimum medium and incubated at room temperature for 20 min. The resulting complex was incubated for 4 h at 37°C with 2 million FADD–/– Jurkat cells per 6 ml of medium in 100-mm dishes. Another 6 ml of medium was added after 4 h, and the complex was incubated for an additional 12 h. The medium was replaced with fresh medium, and the complex was incubated for another 24–36 h. To increase transfection efficiency, a second round of transfection was performed by using TransIT Jurkat transfection reagent (Mirus, Madison, WI) according to manufacturer protocol, and pools of stable cells were then selected as described in refs. 2 and 3 and used for all assays.Antibodies to inhibitor of NF-kB (I-kB)-a, phospho-I-kB-a, and p-FADD were from Cell Signaling Technologies). Antibodies to FADD were obtained from BD Pharmingen and Cell Signaling Technologies. Antibodies to tubulin and actin were purchased from Sigma). Antibodies to cyclins D1 and B1 were purchased from Santa Cruz Biotechnology.
Cloning.
A common 5' primer (5'-GACCGAATTCGCCACCATGGACCCG-TTCCTGGTG-3') was used for cloning FADD and generation of A194 FADD, S194 FADD, and D194 FADD. The 3' primers used for cloning WT FADD and the various mutants of FADD were WT FADD (5'-TGCTCTAGATCATCAGGACGCTTC-GGAGGTA-3'), A194 FADD (5'-GCCTCTAGATCATGCCGCTTCTGCGGTA-GCTGCGTCTGCGTTCCATGCCATCGGTGCCATGGCCCCTGCCCTGTTCTGGA-GGTCAC-3'), D194 FADD (5'-GCCTCTAGATCATGCCGCTTCTGCGGTAGCTGC-GTCTGCGTTCCATGCCATCGGGTCCATGGCCCCTGCCCTGTTCTGGAGGTCAC-3'), and S194 FADD (5'-GCCTCTAGATCATGCCGCTTCTGCGGTAGCTGCG-TCTGCGTTCCATGCCATCGGTGACATGGCCCCTGCCCTGTTCTGGAGGTCAC-3'). All FADD constructs were subcloned into the pEF vector by using EcoRI and XbaI after PCR using pcDNA3-FADD as a template (kindly provided by Arul Chinnaiyan, University of Michigan).Western Blotting Analysis.
The cells in culture dishes were collected and centrifuged at 1,800 × g for 5 min at 4°C. The cell pellet was washed with cold PBS two times and then lysed with a buffer containing 50 mM Tris•HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 50 mM NaF, and 1 mM Na3VO4 and supplemented with complete protease inhibitors mixture (Roche Diagnostics). Cells in lysis buffer were rocked at 4°C for 30 min and sonicated to shear the DNA. The lysates were then cleared by centrifugation. The supernatants collected were estimated for protein content by a detergent-compatible protein assay kit from Bio-Rad. Lysate with equal amounts of protein (either 50, 75, or 100 m g) were separated by Laemmli SDS/PAGE, and the levels of protein expression were detected by Western blot analysis with anti-FADD (1:200 for FADD antibodies from BD Pharmingen, 1:500 for FADD antibodies from Cell Signaling Technologies), p-FADD (1:2,000), I-kB-a (1:1,000), and phospho-I-kB-a (1:500), actin (1:4,000), and tubulin (1:2,000) antibodies. Specific signals were visualized by using the Supersignal West Pico or Femto chemiluminescence detection kits (Pierce).1. Lin, L., Miller, C. T., Contreras, J. I., Prescott, M. S., Dagenais, S. L., Wu, R., Yee, J., Orringer, M. B., Misek, D. E., Hanash, S. M., Glover, T. W. & Beer, D. G. (2002) Cancer Res. 62, 5273–5279.
2. Juo, P., Kuo, C. J., Yuan, J. & Blenis, J. (1998) Curr. Biol. 8, 1001–1008.
3. Juo, P., Woo, M. S., Kuo, C. J., Signorelli, P., Biemann, H. P., Hannun, Y. A. & Blenis, J. (1999) Cell Growth Differ. 10, 797–804.