diagnosis and after acquired EGFR TKI resistance.
0
0
1
CL1-5 si-scramble si-Slug1 si-Slug2 si-Slug3
si-scram
E-cad/TBP ratio (fold to scramble) 0
luminescence units (fold of PC9/gef)
* *
*
*
after the start of treatment
PC9-mock with vehicle control (n=9) PC9-mock with gefitinib treatment (n=9)
PC9-Slug with vehicle control (n=9) PC9-Slug with gefitinib treatment (n=9)
*
- - - + + +
si-scramble si-slug si-Bim slug + si-Bim
B
Online Data Supplement
The Epithelial-Mesenchymal Transition Regulator Slug Confers Resistance to the Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor
Tzu-Hua Chang, Meng-Feng Tsai, Kang-Yi Su, Shang-Gin Wu, Cheng-Po Huang, Sung-Liang Yu, Yung-Luen Yu, Chou-Chin Lan, Chih-Hsin Yang, Shwu-Bin Lin, Chin-Pyng Wu, Jin-Yuan Shih, and Pan-Chyr Yang
Materials and Methods
EGFR Mutation Analysis Using a Sequenom MassARRAY® System
We first performed PCR on the cDNA region corresponding to the EGFR T790M mutation. After the PCR reaction, single-nucleotide extension was performed using a Typlex reagent kit and samples were cleaned up using SpectroClean Resin. Samples were then loaded onto the matrix of a SpectroCHIP® using a Nanodispenser (Matrix) and then analyzed by Bruker Autoflex MALDI-TOF MS. Data were collected and analyzed using Typer4 software (SEQUENOM, San Diego, CA, USA).
Antibodies, Western blotting, and Immunofluorescence Staining
The anti-Slug, anti-caspase 9, anti-bad, anti-puma, and anti-vimentin antibodies were purchased from Abcam (Cambridge, MA). The anti-bim antibody was purchased from Cell Signaling Technology (Danvers, MA). The anti-α-tubulin and anti-lamin B antibodies were purchased from Sigma-Aldrich (Saint Louis, MO). The
anti-E-cadherin antibody was purchased from BD Biosciences (San Diego, CA). The preparation of cells lysates and analysis by Western blotting, and immunofluorescence staining were conducted as described previously (1).
Invasion Assay
The invasion assay was performed using 24-well Costar transwell chambers (Corning, NY) with 8-µm-pore-size membranes coated with a thin layer of BD
Matrigel (BD Biosciences). Cells were seeded onto the Matrigel-coated chamber, incubated for 20 h, and then removed from the upper surface of the filter by scraping with a cotton swab. The cells that adhered to the bottom of the membrane were stained with Giemsa solution.
Transfection of Short Interference RNA
The sequences of short interference RNA (siRNA) duplexes targeting human Slug were listed as below. Sequences of si-Slug 1 were 5’-
GGACACAUUAGAACUCACAdTdT -3’ (sense) and 5’-
UGUGAGUUCUAAUGUGUCCdTdT -3’ (antisense).;
sequences of si-Slug 2 were 5’- CAAACAUAAGCAGCUGCACdTdT -3’ (sense) and 5’- GUGCAGCUGCUUAUGUUUGdTdT -3’ (antisense) ; sequences of si-Slug 3 were 5’-GGACCACAGUGGCUCAGAAdTdT-3’ (sense) and
5’-UUCUGAGCCACUGUGGUCCdTdT-3’ (antisense).
The Slug siRNA was synthesized from Dharmacon (Lafayette, CO). siGENOME SMARTpool reagent for human Slug were obtained from Dharmacon. Transfection was performed using LipofectAMINE 2000 reagent (Invitrogen Carlsbad, California), according to manufacturer’s instructions. Conditions of transfections were optimized for the amounts of siRNA, and LipofectAMINE per well culture as follows: 50 nM of siRNA and 1.5 µL of LipofectAMINE 2000 Reagent. Growth medium were replaced
after 6 hours.
Cloning of stable transfectants
Slug constructions were generated by using the Slug cDNA as described in our previous report (2). Constitutively Slug overexpression clone were performed by introducing Slug plasmid into HCC827 cells by using LipofectAMINE reagent (Invitrogen). The selection of stable transfectant was used 200 μg/mL Gentamicin (G418; Invitrogen) in 3 to 4 weeks. HCC827-Slug9 and HCC827-Slug11 were selected as representatives of Slug overexpression clone. The mock vector-transfected cells (HCC827-mock) were used in bulk for the control.
Slug overexpression in PC9 cells was accomplished by infecting cells with lentiviruses containing the entire human Slug coding region, prepared using the ViraPower Lentiviral Expression System (Invitrogen, Carlsbad, CA), as described by the manufacturer.
Real-time Quantitative RT-PCR
Total mRNA was extracted using TRIzol reagent (Invitrogen). Relative Slug mRNA levels were determined by real-time RT-PCR using the TaqMan EZ RT-PCR Core Reagents and the Applied Biosystems 7500 Real-time PCR System (Applied Biosystems), as previously described (2). Thermal cycling conditions for Slug were 2 min at 50°C, 30 min at 60°C and 5 min at 95°C, followed by 40 cycles of 15 s at
95°C and 1 min at 60°C.
cDNA was synthesized from total RNA by reverse transcription using Superscript II reverse transcriptase (Invitrogen), as described by the manufacturer. Briefly, a reaction mix containing 2 μg total RNA, 50 ng/μl random hexamers, 0.5 mM dNTP mix, 1x First-Strand Buffer, 10 mM DTT, 40 U/μl RNaseOUT, and 200 U/μl
SuperScript II RT was incubated at 65°C for 5 min, chilled on ice for 5 min, incubated at 25°C for 10 min and then at 50°C for 50 min. Relative levels of mRNA of other target were investigated using a SYBR Green dye I-based real-time RT-PCR system.
Reactions were performed on an Applied Biosystems 7500 Real-time PCR System (Applied Biosystems) with SYBR Green PCR Master Mix (Applied Biosystems, CA) using the following cycling conditions: 15 min at 95°C, and 40 cycles of 15 s at 95°C and 1 min at 60°C. At the end of the PCR cycle,melting curve analyses were
performed in order to validate specific generation of the expected PCR product. The Primer sequences are listed in Supplementary Table E1.
Real-time Quantitative PCR
DNA copy number was determined by amplifying 20 ng genomic DNA for
MET and MTHFR (internal control) using the QuantiTect SYBR Green PCR kit (Qiagen) and the Applied Biosystems 7500 Real-time PCR System (Applied Biosystems). Primer sequences for MET copy-number determinations are described in
a previous study (3).
Supplement References:
1. Wang SP, Wang WL, Chang YL, Wu CT, Chao YC, Kao SH, Yuan A, Lin CW, Yang SC, Chan WK, Li KC, Hong TM, Yang PC. P53 controls cancer cell invasion by inducing the mdm2-mediated degradation of slug. Nat Cell Biol 2009;11:694-704.
2. Shih JY, Tsai MF, Chang TH, Chang YL, Yuan A, Yu CJ, Lin SB, Liou GY, Lee ML, Chen JJ, Hong TM, Yang SC, Su JL, Lee YC, Yang PC. Transcription repressor slug promotes carcinoma invasion and predicts outcome of patients with lung
adenocarcinoma. Clin Cancer Res 2005;11:8070-8078.
3. Bean J, Brennan C, Shih JY, Riely G, Viale A, Wang L, Chitale D, Motoi N, Szoke J, Broderick S, Balak M, Chang WC, Yu CJ, Gazdar A, Pass H, Rusch V, Gerald W, Huang SF, Yang PC, Miller V, Ladanyi M, Yang CH, Pao W. Met
amplification occurs with or without t790m mutations in egfr mutant lung tumors with acquired resistance to gefitinib or erlotinib. Proc Natl Acad Sci U S A
2007;104:20932-20937.
Supplementary Figure E1:
Sequencing chromatograms of EGFR T790M. PC9/gef and its subclones (PC9/gef B4 and PC9/gef C4) did not carry the T790M mutation. Arrow indicates the T790M mutation site (nucleotide C to T). H1975 is a positive control. H1650 is a negative control. The experiment was repeated three times with similar results.
Supplementary Figure E2: