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Supplementary Titles and legends to figures
Supplementary Figure S1 Overexpression of FLIP and hnRNP K in NPC
tumor cells. Consecutive NPC tissue sections were stained using anti-FLIP
and anti-hnRNP K antibodies, and were evaluated immunohistochemically. (a)
and (b) Tumor cells with high-level FLIP and hnRNP K expression are shown.
(c) and (d) Tumor cells with relatively weak expression of FLIP and hnRNP K
are shown at 200x magnification. Scale bar, 100 m. The boxed areas are
depicted with a higher magnification (400x) shown as Figure 5 in the text.
Supplementary Figure S2 The effect of hnRNP K knockdown on NF-κB
activation and survival upon TNFα stimulation. (a) hnRNP K knockdown does
not impair nuclear translocation of p65 following TNFα stimulation. NPC-TW02
cells transfected with hnRNP K or control siRNA, followed by treatment with or
without 10 ng/ml TNFα for 30 min. The localization of p65 was visualized by
immunostaining with anti-p65 antibody, and the nucleus location was indicated
by DAPI staining. Scale bar = 20 μm. (b) Nuclear translocation of p65 and p50
analyzed by western blotting. NPC-TW02 cells transfected with hnRNP K or
control siRNA, followed by treatment with or without 10 ng/ml TNFα for 30 min.
Cytoplasmic and nuclear compartments were isolated and the presence of p65,
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p50, IκBα, hnRNP K, tubulin and Sp1 were determined by western blotting.
TNFα stimulation induced nuclear translocation of p65 and p50, as well as
downregulation of IκBα in cells transfected with hnRNP K or control siRNA.
Tubulin and Sp1 were used as loading controls as well as cytoplasmic and
nuclear markers, respectively. (c) hnRNP K knockdown does not affect NPC
cell survival upon TNFα stimulation. The percentage of apoptotic cells of
NPC-TW02 transfected with hnRNP K or control siRNA, followed by treatment
with or without 10 ng/ml TNFα for 48 h, was assessed using Annexin V
staining. All data are presented as the mean ± SD from at least three
experiments.
Supplementary Methods
Cell Culture. The NPC cell lines, NPC-TW01, -TW02 and -TW04, were
maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented
with 10% fetal calf serum, 100 U/ml penicillin, and 100 μg/ml streptomycin at
37°C under 5% CO2.
Affymetrix microarray. RNA samples from NPC tissues and NPC-TW02 cells
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were
isolated
using
TRIzol
reagent
(Invitrogen).
The
biotinylated
oligonucleotide was hybridized to Human Genome U133 Plus 2.0 Array
(Affymetrix, Santa Clara, CA, USA) by the National Yang-Ming University
Genomics Center, Taiwan, following the manufacturer’s instruction. Microarray
data
were
analyzed
using
GeneSpring
software
version
GX
7.31
(Silcogenetics, Redwood, CA, USA). The ratio of the average hybridization
intensity of nine individual NPC tissues and the intensity of one corresponding
pooled adjacent non-tumor tissues was used to represent the relative gene
expression level.
Plasmid Construction. The pGL3-FLIP promoter (-1179 to +281) plasmid
was obtained from Addgene (Addgene plasmid 16016). FLIP-mini promoter
(-37 to +281) plasmid was generated by ligation of the amplified fragment
(using pGL3-FLIP promoter as the template) to KpnI/XhoI-treated pGL3
(Promega). FLIP-mini promoter plasmid carrying R4 mutant were constructed
by using Quikchange Site-Directed Mutagenesis kit (Stratagene) with forward
primer 5’-GGAAC TTGTG TCGAC ATCCA CAGAC GGTTA CTGCA TACTC
AGTC-3’ and reverse primer 5’-GACTG AGTAT GCAGT AACCG TCTGT
GGATG TCGAC ACAAG TTCC-3’. The mutant poly(C) sequences are
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underlined. Flag-tagged FLIP expression plasmid was a kindly gift from Dr. L. J.
Juan (Genomics Research Center, Academia Sinica, Taipei, Taiwan).
Transfection and luciferase assay. NPC-TW02 cells were transfected with 2
μg of pGL3-basic, pGL3-FLIP, pGL3-FLIP-mini, or pGL3-FLIP-mini mutant
plasmid and 10 ng of pRL-CMV (Promega) by using Lipofectamine (Invitrogen),
according to the manufacturer’s instructions. Following 24 hours, the luciferase
activities were measured using the Dual Luciferase Reporter Assay System
(Promega). Firefly and Renilla luciferase activities were measured using the
Dual-Glo Luciferase Assay System (Promega) according to the manufacturer's
instructions. Firefly luciferase activities were standardized to Renilla activities.
Each bar represents an average of at least three independent experiments.
Error bars are presented as standard deviations using Microsoft Office Excel.
Apoptosis analysis. Apoptosis induced by the human recombinant TRAIL
and TNFα (PeproTech) was determined by measuring the population of
Annexin V–positive cells. Cells were treated with TRAIL to induce apoptosis.
Following incubation for 24 h, phosphatidylserine externalization was analyzed
using the Vybrant® Apoptosis Assay Kit #2 (Invitrogen). Cells (2 x 105) were
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Chen et al.
washed with PBS, and adjusted in 1x binding buffer to a concentration of 2 x
106/mL. Alexa Fluor® 488 annexin V (5 μ
μl of the cell
suspension, and incubated for 15 min at room temperature. Samples were
analyzed (10,000 events) using a FACS Caliber Flow Cytometer (Becton
Dickinson).
Immunoprecipitation and Western blotting. For isolating nuclear extracts of
NPC-TW02 cells, the Compartmental Protein Extraction Kit (Millipore) was
used following the manufacturer’s instructions. Immunoprecipitates were
precipitated from 200 μg of nuclear extracts using specific antibodies against
hnRNP K (Invitrogen Life Technologies), AP-2α
(Santa Cruz Biotechnology),
or nucleolin (a kindly gift from Dr. N. H. Yeh, Institute of Microbiology and
Immunology, National Yang-Ming University, Taipei, Taiwan). Mouse and
rabbit IgG (Millipore) was used as a negative control. The resulting products
were eluted with 2X SDS sample buffer and analyzed by Western blotting.
Proteins of interest were detected with specific antibodies and an enhanced
chemiluminesence system (Amersham Pharmacia). The utilized antibodies
were obtained as follows: Antibodies including anti-hnRNP K, anti-hnRNP F,
anti-p300, anti-YB-1, anti-DR4, anti-YY1, anti-p65, anti-IκBα, and anti-Sp1
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were purchased from Santa Cruz Biotechnology; anti-FLIP from Alexis
biochemicals; anti-DR5 from ProSci, Inc.; anti-p50 from Millipore and
anti-tubulin and anti-actin from MDBio Inc. The anti-AP-2α and anti-nucleolin
were the same ones used for immunoprecipitation.
Immunofluorescence microscopy. NPC-TW02 cells were grown on glass
coverslips for 24 h, followed by transfection of hnRNP K or control siRNA, and
then the treatment with or without 10 ng/ml TNFα for 30 min. Endogenous p65
was detected with a specific antibody, and sequentially incubated with a
fluorescein isothiocyanate-conjugated secondary antibody (BD Transduction
Laboratories,
Lexington,
KY,
USA).
Nuclei
were
stained
with
4’-6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, Poole, UK) during the
secondary antibody incubation. The localization of p65 was observed by
confocal fluorescence microscopy with a Leica TCS SP2 confocal laser
scanning microscope (Leica Microsystems Inc.).
Peptide labeling and fractionation by offline 2D liquid chromatography
(2D-LC). One hundred micrograms of extracted proteins for each sample was
enzymatically cleaved with trypsin, reduced and blocked with 5 mM TCEP and
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10 mM MMTS, and then labeled using isobaric reagents (iTRAQ™ Reagents
Multiplex Kit, Applied Biosystems) according to the manufacturer's protocol
(Applied Biosystems). All labeled extracts were combined and then
fractionated on Germini C18 column (Phenomenex, 3x100 mm) coupled to a
Breeze HPLC instrument (Waters) and equilibrated with buffer A (water at
pH10.0 by ammonia). The peptides were eluted using a segmented gradient
from 2 % to 100 % solvent B (100 % acetonitrile, pH 10.0 by ammonium) over
80 min.
Shotgun quantitative proteomics on LTQ-Orbitrap mass spectrometer.
NanoLC−nanoESi-MS/MS analysis was performed on a Surveyor nanoflow
system connected to an LTQ-Orbitrap hybrid mass spectrometer (Thermo
Fisher, San Jose). Peptide mixtures in solvent A (0.1% formic acid) were
loaded onto a PicoFrit BioBasic C18 capillary column (75 μm × 150 mm, New
Objective, Woburn, MA) and separated using a segment gradient elution from
2 % to 100 % of solvent B (100 % acetonitrile with 0.1% formic acid) over 90
min. The MS survey scans for all experiments were acquired in the Orbitrap
recording a window between 430 and 2000 m/z. The resolution was set to
30000, and automatic gain control (AGC, target at 10 6) was used to prevent
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over-filling of the ion trap. Up to four most intense ions with minimal signal
intensity of 20000 were sequentially isolated for MS/MS fragmentation in the
order of intensity of precursor peaks. The MS/MS spectra were detected in the
linear ion trap with previously selected ions dynamically excluded for 120 s and
collected by Pulsed Q dissociation (PQD) method under the control of Xcalibur
2.0 software with the following settings: normalized collision energy at 30 %, Q
activation at 0.7, the activation time of 0.1 and the isolation width at 2.0. The
maximum fill time for each MS and MS/MS scan was 1 s and 100 ms,
respectively. Four MS/MS microscans were averaged to create proper signal
intensity for peptide quantification using iTRAQ reporter ions. Ions with singly
and over four charge states were excluded.
LC-MS/MS data analysis. The MS and MS/MS raw data were converted to
MGF peak list files by DTASuperCharge 1.37 using SmartPicking with 200
segment size (Th) and max search level of 8. The generated MGF files were
searched against a SwissProt human database (v. 56; 20333 sequences),
using an in house Mascot Daemon 2.2.2 server. Search criteria were defined
as trypsin digestion, fix modification of methylthio (C), variable modifications of
N-terminal acetylation of protein, conversion of N-terminal glutamine to
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pyro-glutamate, oxidation (M), and iTRAQ labeling at K residue and
N-terminus of peptides. One missed cleavage, mass accuracy of 6 ppm on the
parent ion and 0.6 Da on the fragment ions were allowed. This protein
identification results were further filtering using an open-source proteomics
statistical analysis software (Trans-Proteomic Pipeline, TPP v4.2 JETSTREAM
rev 1, http:// tools.proteomecenter.org/TPP.php) developed at the Institute for
Systems
Biology
(Seattle,
WA),
with
both
of
PeptideProphet
and
ProteinProphet probability cutoffs set at 0.95. The filtered protein hit dataset fit
to a 99.8 % sensitivity and an estimated false positive error rate of 0.003% in
this study. Quantitative information was simultaneously obtained using the
Libra program which is one component of TPP.
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