1/7/2013
Supplemental Information
Aberrant IKKα and IKKβ cooperatively activate NF-B and induce EGFR/AP1
signaling to promote survival and migration of head and neck cancer
Liesl K. Nottingham,1,2 Carol H. Yan,1,2,3 Xinping Yang,1 Han Si,1 Jamie Coupar,1
Yansong Bian, 1 Tsu-Fan Cheng,1 Clint Allen,1,4 Pattatheyil Arun,1 David Gius,1,5 Lenny
Dang,1,6 Carter Van Waes,1,7 and Zhong Chen1,7
Supplemental Materials and Methods
Immunohistochemical analysis of HNSCC tissue specimens
Frozen tissue samples of HNSCC from the Cooperative Human Tissue Network (CHTN)
were obtained and sectioned. Detailed procedures for immunohistochemistry (IHC) and
the histoscore were previously described (1), which combine both staining intensity and
percentage cells stained. Rabbit anti-IKK (#2682) and Phospho-IKK (Ser176/180,
#2697) were purchased from Cell Signaling. Rabbit anti-IKK (#ab6146) was purchased
from Abcam. Mouse anti-human pan cytokeratin (IgG1, 1 μg/ml, Novocastra Lab,
Newcastle upon Tyne, UK). Histology images were taken under microscope at 100X
(low) and 400X (high) magnifications.
Cell lines and culture
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University of Michigan head and neck squamous cell carcinoma (UM-SCC) cell lines 1,
6, 9, 11A, 11B, 22A, 22B, 38, and 46 were a generous gift from T.E. Carey (University
of Michigan, Ann Arbor, MI). Cells were maintained in Eagle’s Minimal Essential Media
(Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum,
penicillin, streptomycin and L-glutamine (complete media) under standard growth
conditions (37°C, 5% CO2, humidified atmosphere). Cell DNA was sent for sequence
genotyping in 2008 and fall 2010 to compare and verify their unique origin from original
stocks, as recently described (23). The 9 loci analyzed included D3S1358, D5S818,
D7S820, D8S1179, D13S317, D18S51, D21S11, FGA, vWA and amelogenin. Human
epidermal keratinocytes were obtained from (Invitrogen, Carlsbad, CA) and harvested at
less than passage 5.
siRNAs and plasmids
Multiple siRNAs targeting IKK or IKK were obtained from IDT (Coralville, IA), and
the knockdown efficiency was tested individually. The three most effective duplexes of
siRNAs for each target were pooled for the experiments. Cy3™ DS transfection control
and DS Scrambled Neg siRNA (IDT) were used as controls. IKK expression and mutant
constructs were kindly provided by Dr. Ulrich Siebenlist (NIH/NIAID, Bethesda, MD)
and Dr. Michael Karin (UC San Diego) consisting of IKKα and β wild type (WT),
phosphoacceptor-mutant (SS->AA), constitutively activated (SS->EE), and kinase dead
(K44A) vectors. The functional domains were depicted in Fig 2A, upper panels. 5XNFkB-luciferase and 7XAP1-luciferase reporter gene constructs were purchased from
Stratagene. The luciferase plasmids containing IL-8 promoter sequence (-133~+44bp
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from TSS) with point mutations were kindly provided by Dr. Naofumi Mukaida (2). The
IB-Photinus luciferase reporter was kindly provided by the laboratory of Dr. Louis M.
Staudt, and the IB luciferase fusion protein serves as an indicator of IKK activity (3).
To determine the effect of kinase knockdown, UM-SCC1 cells were co-transfected with
IKK or IKK specific siRNA and the reporter plasmid. Two concentrations (30nM and
60nM) of control siRNA were used to match the siRNA concentration in the single or
dual knockdown, respectively. No statistical difference of reporter activity was observed
between the two controls, so only one control was presented.
Western blot
11A cells were plated in 10cm dishes, 1x106 cells per plate. After 24 hours cells were
transfected with Control siRNA, IKKα siRNA, IKKβ siRNA, or both IKKα and IKKβ
siRNA. Control siRNA had a final concentration of 60nM and both IKKα and IKKβ total
siRNA concentration was 30nM (10nM of each duplex). 47 hours post transfection, cells
were treated with TNF- (10ng/ml) for 1 hours before collecting protein lysates. Or 44
hours post transfection, cells were treated with lymphotoxin α1/β2 (100ng/ml) for 4 hours
before collecting nuclear and cytoplasmic protein lysates. For testing AP1 protein
expression, whole cell lysates were collected after knockdown of IKKs for 48 hours.
Whole cell, nuclear, and cytoplasmic lysates were obtained using a Nuclear Extraction
Kit from Active Motif (Carlsbad, CA). 15μg of lysates were loaded per sample and
analyzed as previously described (4). Ponceau-S staining was used to visualize total
protein in blots using nuclear lysates. Antibodies used in these studies (Cell Signaling
Technology, Danvers, MA) included IKKβ (2684), β-actin (4967), phospho-IKKα/β
3
(2697), RelB (4954), Oct1 (4428), NFB2 p100/p52 (4882), EGFR (4405), Fra1 (5281),
JunB (3746), cJun (9165), and anti-rabbit IgG–HRP (7074) as the secondary antibody.
Anti-IKK antibody was obtained from Imgenex (San Diego, CA) and anti-p65 (sc8008) from Santa Cruz Biotechnology (Santa Cruz, CA). The rabbit anti-S222-p52
phospho-specific antibody was kindly provided by Dr. Neil Perkins, who developed this
antibody in Biogenes, using the peptide CIHDSK-pS-PGASN-amide as the immunogen
(5). The Western blot protocol was conducted as described previously in 4oC with 5%
milk in TBST (0.1% tween) buffer (6).
Reporter gene assay
UM-SCC cells were co-transfected with 0.15 µg/well of 5X-NF-κB (Stratagene, Cedar
Creek, TX) or IκBα-luciferase reporter and 0.003 µg/well of pRSV-LacZ (ATCC) using
Lipofectamine2000 in Opti-Mem I (Invitrogen, Carlsbad, CA) with the addition of IKK
plasmids at 0.2µg/well per specific IKK subunit or siRNA at 30nM per subunit (IDT,
Coralville, IA). At 24 and 48 hrs, reporter gene activity was assayed using the Dual-Light
Luciferase Reporter Gene Assay System (Tropix, Bedford, MA), using the Wallac
VICTOR2 1420 Multilabel Counter (Waltham, MA).
Each sample was assayed in
triplicate and data were presented as the mean + standard deviation (SD).
MTT assay
UM-SCC cells were plated in 100mm plates, transfected with si-IKKα, -IKKβ, DS
scrambled negative control siRNA or Lipofectamine2000 control and grown for 48 hours
before trysinized and counted for a 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium
4
bromide (MTT) assay. Cells were plated in sextuplicate onto a 96-well plate at 3 × 103
cells/well in 100 μL of complete MEM. Cell proliferation was measured every subsequent
day for 5 days using a MTT Cell Proliferation kit (Roche Diagnostics). The absorbance was
measured by a μQuant microplate reader (Bio-Tek Instruments) at 570nm wavelength.
Wound healing assay
A wound closure assay was performed after scratching confluent monolayers of UMSCC1 transfected with negative control siRNA (30nM and 60nM), siIKKα (30nM),
siIKKβ (30nM), or siIKKα + siIKKβ (60nM) in 6-well plates for 48 hrs before two cellfree perpendicular scratches were created across the midline of each dish with a 1000uL
pipette tip. The scratched areas were photographed after adding fresh medium at 0hr,
14hrs, 20hrs and 30hrs with an Olympus IX70 inverted microscope and a Canon camera.
The distance was measured at twelve preset points along the wound for each sample and
averaged and closure was quantified by NIH ImageJ 1.37V software. The distance of the
wound closure was quantified and statistical analysis was performed as the percent
closure relative to the width of the original scratch by student’s t-test. As there was no
statistical significance of the wound healing in cells transfected with negative control
siRNA at either 30nM or 60nM concentration, only one control was presented.
Supplemental Figure Legends:
Supplemental Figure 1 Down regulated gene expression after IKK single or dual
knockdowns by siRNA in UM-SCC1 cells. UM-SCC1 cells were transfected with
5
siRNAs targeted IKKα alone (A), IKKβ alone (B), or IKKα & IKKβ in combination (C).
The gene expression profiles were compared between knockdown samples with control
scramble siRNA transfected cells, and (D) represented decreased gene expression
observed in all experimental conditions. The decreased gene expression was presented as
the negative fold change compared to controls.
Supplemental Figure 2 Dual IKK and  chemical inhibitors exhibit greatest
inhibition of UM-SCC cell proliferation. Cell proliferation was measured in a 5 day
MTT assay in UM-SCC1 and UM-SCC11B lines treated with various concentrations of
chemical inhibitors. Cell growth rates were analyzed in sextuplicate.
Supplemental Figure 3 Drugs targeting IKKα and β promote apoptosis and cell
cycle arrest of UM-SCC cells. A, Flow cytometry was performed to test cell-cycle and
DNA fragmentation in sub-G0/G1 phase (% cell death) of UM-SCC1 treated with SC514 (50M, left) and UM-SCC11B treated with Wedelolactone (20M, right) for 24hrs.
B, The effects of drugs treated for 24hrs (same concentrations as in A) on cell
morphology of UM-SCC1 and UM-SCC11B by micrographs (100X).
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