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Supplementary Materials
Tissue microarrays. Tissue microarrays were constructed with cores from
formalin-fixed, paraffin-embedded blocks. Triplicate core samples were included for each
tumor. All samples were obtained retrospectively from 252 patients with NSCLC treated
at The University of Texas M. D. Anderson Cancer Center. Only patients for whom
signed informed consent and pertinent clinical data were available were included. These
studies were submitted as a protocol to the IRB at M. D. Anderson Cancer Center, and
the protocol was approved prior to their initiation.
Mutational analysis of tumor samples. Approximately 103 tumor cells were
microdissected from sequential, 8 m-thick tissue sections that were stained with H&E,
fixed in formalin, and embedded in paraffin. DNA was extracted from tumor samples
using 25 l of Pico Pure DNA extraction solution (Arcturus) containing proteinase K and
incubated at 65 °C for 24 h. Exons 18 to 21 of EGFR and codon 12 of KRAS were
amplified by PCR using intron-based primers. All PCR products were directly sequenced
using the Applied Biosystems PRISM dye terminator cycle sequencing method (PerkinElmer). All sequence variants were confirmed by independent PCR amplifications from
at least two independent microdissections and were sequenced in both directions.
Immunohistochemistry. We used a DakoCytomation dual-polymer
immunohistochemistry system (Carpinteria, CA). After the tissue sections were baked
and deparaffinized, the slides were rinsed three times with MilliQ water. Antigens were
retrieved by steaming the slides in Dako target retrieval solution for 25 min. Endogenous
peroxidase was blocked with 3% hydrogen peroxide in water for 5 min. The slides were
rinsed with MilliQ water and incubated in TBS-20 for about 10 min. After being blocked
with Dako serum-free protein block for 20 min, the slides were incubated with a 1:50
dilution of polyclonal rabbit antibody to p-JNK (Cell Signaling) overnight at 4ºC in 1%
bovine serum albumin in TBS. The slides were then rinsed and incubated with Dako
EnVision+ Dual Link reagent for 30 min. The slides were stained with three to four drops
of diaminobenzidine solution and counterstained with Mayer hematoxylin for 1 min. After
being rinsed and incubated with Scott’s bluing reagent, the slides were dehydrated and
mounted using a xylene-based mounting medium and a cover slip.
Two pathologists (I.W. and M.S.) independently quantified p-JNK
immunostaining. p-JNK staining was quantified based on the percentages of cells
staining positively in nuclear and cytoplasmic compartments. For each tumor, the p-JNK
score was defined as the average of the three core samples, with separate
determinations of nuclear and cytoplasmic scores. Nuclear scores were determined from
a minimum of 200 cells per tumor. A tumor was considered positive if at least 10% of the
cells examined stained positively in either compartment.
Generation of stable transfectants from retroviruses. Constitutively active
human MKK4 (S287E/T291D mutant) cDNA (MKK4-ED) in a pEBG vector was a gift
from Dr. John M. Kyriakis (Harvard University, Boston, MA). A doxycycline-inducible
retroviral gene expression system (BD Biosciences) was used to inducibly express
MKK4-ED in BEAS-2B and HB56B cells. A Flag-tagged MKK4-ED construct was created
by PCR using the pEBG-MKK4-ED vector as a template. The resulting fragment was
subcloned into the HindIII and BamH1 sites of a vector containing a tetracyclineresponsive element (TRE), with hygromycin as the selection marker, to create the TREMKK4-ED plasmid. As a negative control, the firefly luciferase gene was subcloned into
the TRE vector. For virus production, vectors containing TRE-MKK4-ED, TRE-luciferase,
and the tetracycline-controlled transactivator (rtTA) were transfected into PT67 cells
using Lipofectamine Plus (Life Technologies, Rockville, MD) following the manufacturer's
instructions. After antibiotic selection, the viral supernatant was recovered from the mass
transfectants, filtered, quantified, and stored at –80C for future infections.
BEAS-2B and HB56B target cells were infected with the rtTA retroviral
supernatant using 8 g/ml Polybrene. The cells were selected with 50 g/ml G418 after
48 h, and single-cell subclones were isolated after 14 days. Clones expressing rtTA
were subjected to transient transfection with TRE-luciferase and then lysed, and
luciferase was quantified by reporter assays. One clone expressing high levels of rtTA
was then chosen for infection with TRE-MKK4-ED or TRE-luciferase retroviral
supernatants as described above. The cells were selected in 250 g/ml hygromycin, and
single-cell subclones were isolated and expanded for characterization.
The JNK1/2 short hairpin RNA (shRNA; 5-AAA GAA UGU CCU ACC UUC UTT3 and 5-AGA AGG UAG GAC AUU CUU UTT-3) (Geqiang et al., 2004) targeting a
common sequence 377 nucleotides downstream from the start codon in JNK1 and JNK2
mRNA was synthesized and subcloned into the p-Super retroviral vector (OligoEngine,
Seattle, WA). The subsequent vector was then transfected into the packaging PT67
cells, and virus was produced. BEAS-2B double transfectants (rtTA/MKK4-ED) were
infected with the JNK1/2 shRNA retroviruses and selected with 2.5 g/ml puromycin.
Single-cell subclones were isolated and expanded for characterization.
Immunoblotting. BEAS-2B and HB56B transfectants were treated with or
without doxycycline for 36 h and then subjected to western blotting. To examine the role
of JNK in C8 cells, SP600125 was added 1 h prior to lysis. NSCLC cell lines were
cultured in serum-free conditions for 24 h prior to lysis. Cells were washed twice with
PBS and lysed in lysis buffer containing 20 mM Tris (pH 7.4), 150 mM NaCl, 1 mM
EGTA, 1 mM EDTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM sodium glycerophosphate, 1 mM sodium vanadate, 1 g/ml leupeptin, and 1 mM
phenylmethylsulfonyl fluoride. Insoluble material was removed by centrifugation at
10,000g at 4C for 10 min. Protein concentration in the supernatants was measured
with a Quick Start Bradford dye kit (Bio-Rad Laboratories, Hercules, CA). Equal amounts
of protein (50-100 g per lane) were separated in 10-12% of SDS-polyacrylamide gels
and transferred to polyvinylidene fluoride membranes (Bio-Rad Laboratories). The blots
were blocked in PBS with 0.05% Tween-20 and 5% nonfat dry milk for 1 h and incubated
overnight at 4C with primary antibodies per the manufacturer's recommendations.
Proteins were visualized by enhanced chemiluminescence (Amersham,
Buckinghamshire, UK).
Comparative genomic hybridization microarrays. Comparative genomic
hybridization array analysis was performed on NSCLC cell lines as previously described
(Pollack et al., 1999; Pollack et al., 2002) using 4 g of genomic DNA to examine copy
numbers of MKK4 and cJun gene loci on chromosomes 17q12 and 1p32.1, respectively.
Analysis was done using Visual Basic software (MATRIX 1.30), which sorts array data
according to chromosome location (based on Golden Path release hg17
(http://genome.ucsc.edu) and calculates five-point moving averages to reduce local
variations. The program then generates a color display, with red for DNA gain and green
for DNA loss.
Proliferation assays. Cell proliferation was measured using the 3-(4,5
dimethylthiazol-2-thiazyl)-2,5-diphenyl-tetrazolium bromide (MTT; Sigma) colorimetric
dye reduction method. Cells (104) were cultured in a 96-well plate. NSCLC cell lines
were treated for 72 h with or without SP600125. BEAS-2B and HB56B transfectants
were treated for 24 h with or without 0.5 g/ml doxycycline. To examine the role of JNK
in C8 cells, SP600125 was added 12 h prior to lysis. At the completion of treatment, 3
mg/ml MTT reagent was added to the medium, and the cells were incubated for an
additional 30 min. The medium was replaced with 200 l of dimethylsulfoxide, and the
absorbance of each well was measured at 570 nm with a micro-ELISA reader.
Invasion and migration assays. In vitro invasion assays were performed using
24-well Matrigel invasion chambers (BD Biosciences) in which the upper compartment of
the chambers was coated with Matrigel. The lower compartment was filled with normal
growth medium for HBE cells. Cells were placed in the upper compartment in medium
with or without doxycycline and incubated for 24 h. The medium was aspirated after the
incubation, and the cells were removed from the upper compartment by scrubbing with a
cotton swab. The lower compartment was stained with SureStain WRIGHT (Fisher
Diagnostics, Middleton, VA) and then washed with water. The invasive cells (those on
the lower side of the membrane) were counted by microscopy at 20 magnification.
Mean values were calculated from cell counts in five random fields for each filter. The
assay was performed three times, and results from a single representative experiment
were illustrated.
In vitro migration assays were similarly performed using a 24-well Transwell unit
with polycarbonate filters. The filters were not coated with Matrigel for the migration
assays.
Wound-healing assays. Cells were cultured as monolayers in a six-well plate.
Once the cells reached 90% confluency, a 2-mm-wide wound was made along the
plastic surface with a pipette tip. The cells were washed twice with PBS, and fresh
medium with or without doxycycline was added to the cells. The cells were allowed to
migrate for 3 d, and photographs were taken at the indicated time points.
Anchorage-independent growth assay. Soft-agar growth assays were
performed in six-well plates. Cells (8  103) suspended in 0.5% soft agar, with or without
doxycycline, were layered on top of 1% low-melting-point agarose (SeaPlaque, FMC,
Rockland, ME) in complete medium. The plates were cultured at 37C in the presence of
5% CO2 for 10 days and then photographed. The number of colonies in each well was
determined by microscopy at 20 magnification.
Tumorigenicity assay. C8 cells, S1 cells, or luciferase controls were injected
(5x106 cells per injection) into the flanks of nude mice (Charles River Laboratories,
Wilmington, MA). There were four treatment cohorts (5 mice per cohort, 20 mice total).
Three cohorts (C8, S1, and luciferase controls) were treated with doxycline (2 mg/ml) in
the drinking water. A fourth cohort (luciferase controls receiving no doxycycline) was
included as a control. Mice were observed for 21 days for evidence of tumor formation.
Hoechst staining. Apoptosis was measured by Hoechst staining. Cells were left
untreated or treated with SP600125 for 36 h and then fixed with 4% paraformaldehyde at
room temperature for 20 min. As a positive control, H2009 NSCLC cells were treated for
3 h with staurosprine (2 g/mL). Hoechst 33342 (Sigma) was added directly to a final
concentration of 10 g/ml. Cells were stained in the dark at 4C overnight and examined
with an Olympus IX71 fluorescence microscope the next day.
Kinase assays. JNK kinase activity was analyzed using a nonradioactive stressactivated protein kinase/JNK kinase assay kit (Cell Signaling Technology). Briefly, cells
were lysed in the cell lysis buffer provided by the manufacturer. After centrifugation at
12000  g for 10 min, supernatants were incubated with an N-terminal c-Jun fusion
protein bound to glutathione-Sepharose beads to pull down the JNK. The beads were
washed twice to remove nonspecific proteins. The kinase reaction was performed in the
presence of ATP in 25 mM Tris-HCl (pH 7.5), 5 mM -glycerophosphate, 2 mM
dithiothreitol, 0.1 mM sodium vanadate, and 10 mM MgCl2. JNK-induced
phosphorylation of c-Jun was measured by immunoblotting with antibody against p-cJun.
Similarly, p38 kinase activity was examined using a nonradioactive p38 kinase
assay kit (Cell Signaling Technology). Briefly, cell extracts were incubated overnight with
immobilized monoclonal antibody against p-p38. The kinase reaction was performed in
the presence of 100 µM cold ATP and 2 µg of ATF-2 fusion protein. Phosphorylation of
the ATF-2 fusion protein at Thr71 was measured by western blotting with antibody
against p-ATF-2.
Statistical analysis. For analysis of NSCLC biopsy samples, data were
summarized using standard descriptive statistics and frequency tabulation. Associations
between categorical variables were assessed by cross-tabulation, chi-squared test, and
Fisher’s exact test. Associations between pairs of numerical variables were assessed by
Spearman rank-sum test. Kruskal-Wallis and Wilcoxon rank-sum tests were performed
to assess the associations between p-JNK and patients’ clinicopathologic features.
Univariate Cox proportional hazards regression models were used to assess the
effect of JNK and patients’ characteristics on overall and recurrence-free survival.
Overall survival was defined as the time from operation to death from any cause.
Recurrence-free survival was defined as the time from operation to recurrence or last
recurrence-free follow-up examination. Survival curves were estimated using the
Kaplan-Meier method.
Two-way analysis of variance was used to analyze the MTT assays, and t-tests
were used to analyze the other in vitro assays. P values of less than 0.05 were
considered significant. All computations were carried out using SAS (Cary, NC) and Splus (Cambridge, MA) software.