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Supplementary Materials and Methods
Western blot and cytoplasmic/nuclear extraction
Cells were lysed with the buffer containing 10% glycerol, 50mM Tris pH 7.7,
150mM NaCl, 0.5% NP40 and a cocktail of protease (Sigma P8340) and phosphatase
inhibitors (Sigma P2850 and P5726). Electrophoresis and Western blot procedures were
performed as previously described (Yang et al., 2007b) using dilutions of antibodies
recommended by the manufacturers. Experiments were repeated at least three times,
blots were scanned for band density, and mean density values were quantified using
ImageJ (NIH). Cytoplasmic/nuclear extraction was performed as previously described
(Yang et al., 2007b).
Antibody information
Primary antibodies used are NIK (Novus NB120-7204), p100/p52 (Upstate 05361), RelA (Santa Cruz sc-109) phospho-p65 (Ser536) (Cell Signaling 3033S), IB (BD
Transduction Laboratories 610690), BCL2 (Santa Cruz sc-7382), Survivin (Cell Signaling
2808), cIAP1 (Alexis: ALX-803-335), BCL-XL (Santa Cruz sc-634), cFLIP (Cell Signaling
3210), -catenin (Abcam ab2365), GAPDH (Cell Signaling 4777), lamin A/C (Cell
Signlaing 5174), actin (Santa Cruz sc-1616 or 1616R) or tubulin (Sigma T2220).
Secondary antibodies are (Molecular Probes Alexa Fluor 680 or 800 A21058/ A21084;
Rockland 611-732-127 or Jackson Laboratories 115-035-146 (goat anti-mouse), 705035-003 (donkey anti-goat), 111-035-144 (goat anti-rabbit).
IKK kinase assay
Non-silencing or knock-down cells were starved overnight and treated with
vehicle or TNF (20ng/ml) (PreproTech 300-01A) for 30 min. Using 200 µg of
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cytoplasmic extract prepared from untreated or treated cells, IKK kinase assay was
performed as previously described (Yang et al., 2007b).
Quantitative Real-time PCR
RNA was isolated from non-silencing and knock-down cells using RNeasy kit (Qiagen
74104) according to the manufacturer’s protocol. Then, cDNA was synthesized from one
µg of RNA using iScript cDNA Syn RT-PCR kit (BioRad 1708890) according to the user
manual. The resulting cDNA was then amplified by quantitative real-time PCR using IQ
Real Time Sybr Green PCR supermix (BioRad 1708880) and BioRad IcyclerIQ
Multicolor Real-Time PCR Detection System. cDNA levels were normalized to actin that
was amplified with the following primers: 5’-caccacaccttctacaatgag-3’, and 5’atagcacagcctggatagc-3’.
NIK primers are: 5’tgcggaaagtgggagatcctgaat-3’, and 5’-
tgtactgtttggacccagcgatga-3’. For Bcl2, cIAP1, survivin, Axin2, Tcf7 and genes from
microarray analysis, primers were purchased from SABiosciences. Data are shown as
fold changes in NIK knock-down cells compared to the non-silencing control, and each
gene was normalized to the loading control (actin or tubulin). Statistical significance was
determined by calculating 95% confidence interval.
Chromatin Immunoprecipitation (ChIP)
Chromatin immunoprecipitation was performed following the protocol using
Simple Enzymatic ChIP kit (Cell Signaling #9002). Briefly, cells were cross-linked using
1% of paraformaldehyde, permeablized and chromatin was digested with microccocal
nuclease at 37ºC for 20min. Chromatin was immunoprecipitated using IgG, histone, p65
(sc-109x) or -catenin (ab2365) antibodies for 2 h at 4ºC. Immunoprecipitated chromatin
was purified and PCR was performed using the following primers amplifying the survivin
promoter: 5’-GGGGCGCTAGGTGTGGG-3’, 5’-TTCAAATCTGGCGGTTAATGGC-3’ (-
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catenin_human);
5’-CTTCTGCTTCCCTTCCAACC-3’,
5’-
GCCTTGCGCAAGGGTGTGATT-3’ (-catenin_mouse). PCR was performed with
annealing temperature 55-60 ºC and with cycle 30-35. Experiments were performed at
least three times.
Cell cycle analysis
Non-silencing and NIK knock-down cells were synchronized by thymidine block
(2mM for 16h). After indicated amount of time of release from thymidine block, cells were
collected, fixed with 70% EtOH and stained with PI staining buffer (0.1% Triton X-100,
0.2mg/ml of RNAse, 20g/ml of PI, 1mM of EDTA) for 30min at RT, followed by flow
cytometry analysis. Experiments were performed at least three times and statistical
significance was determined by using t-test. Significance was assumed if the p-value
0.05.
Immunofluorescence
Cells on coverslips were fixed with 4% paraformaldehyde and permeabilized with
0.2-0.5% of Triton-X 100 in 1xPBS for 5min. Then cells were blocked with 10% normal
donkey serum in 1xPBS for 30 minutes and incubated with β-catenin antibody (abcam
ab2365, 1:100) for 2h at room temperature (RT), followed by three washes with 0.1%
Tween-20 in 1xPBS (1xPBST) and incubation with secondary antibody conjugated with
Cy2 (Jackson ImmunoResearch 715-225-150, 1:100) for 2 h at RT. Then, slides were
washed three times and stained with Hoechst nuclear stain during the last wash.
Coverslips were then air dried and mounted onto glass slides. Images were taken using
a 63x oil lens Plan-Apochromat on Zeiss Axioplan 2 microscope and Hamamatsu Orca
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ER fluorescence camera. Images were pseudo-colored and processed using Metamorph
Software (Molecular Devices).
Immunofluorescence staining of Tissue Microarray (TMA)
Human patient samples for TMA were collected according to the protocol
approved by Institutional Review Board of Vanderbilt University. Each sample was
spotted in triplicates or duplicates. Tissues were sectioned to 4m thickness and
immunofluorescent staining was performed. NIK antibody in immunofluorescent staining
was first validated in human melanoma xenograft from WM115 cells with or without NIK
knock-down. After validation, paraffin embedded nevus and melanoma tissues were
deparaffinized by washing two times in Xylene for 10 min each wash, followed by
washing in a series of ethanol dilutions (100%, 90%, 80%, 70%, 50% ethanol). Then
tissues were permeabilized in 1xTBST (0.05% Tween-20) for 15min, washed with
1xTBS and rinsed with dH2O. Antigen retrieval was performed by boiling tissue slides in
10mM sodium citrate pH 6.0 for 12 min. After the slides were cooled down, tissues were
blocked with the blocking buffer containing 10mM Tris-HCl pH 7.4, 0.1M MgCl2, 0.5%
Tween-20, 1% BSA and 5% goat serum. Tissues were incubated with NIK antibody
(Novus 120-7204, 1:100) in the blocking buffer overnight at 4C. Slides were then
washed with 1xTBST for 3x10min each and incubated with Alexa Flour 488 goat antirabbit secondary antibody (Invitrogen A-11008, 1:1000) for 2 h at RT. Slides were
washed again with 1xTBST for 3x10 min each, counterstained with Hoechst and
mounted with Prolong Gold antifade reagent (Invitrogen P36930). Images were scanned
and quantitated using Ariol SL-50 imaging system (Genetix). Quantitative data was
presented as (area of NIK staining/area of Hoechst staining)x100. Damaged tissue cores
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were excluded from quantitation. Statistical significance was determined by using
ANOVA. Significance was assumed if the p-value 0.05.
Immunohistochemistry
Five micron sections were placed on charged slides, paraffin removed by xylene
and ethanol washes, and tissue rehydrated.
For caspase-3 staining, endogenous
peroxidase was neutralized with 0.3% H2O2 for 20 min followed by Ultra V block for 5
min (Labvision) for nonspecific staining blocking.
For Ki-67 staining, sections were
rehydrated and placed in heated Target Retrieval Solution (Labvision). Endogenous
peroxidase was neutralized with 0.03% H2O2 followed by a casein-based protein block
(DakoCytomation) to minimize nonspecific staining. Then, sections were incubated with
rabbit anti-human cleaved caspase-3 (Promega) or Ki-67 (Vector Laboratories VP-K451)
for 60 min. The Dako Envision+ HRP/DAB+ System (DakoCytomation) was used to
produce localized, visible staining. The slides were lightly counterstained with Mayer’s
hematoxylin, dehydrated and coverslipped.
Microarray
Affymetrix WT Sense reactions, using the Affymetrix WT Sense reaction kit
(#900652), were assembled and run following Affymetrix protocol. Briefly, a sample mix
of 200ng total RNA, and poly-A spike in controls (included in kit), were brought to a 5 l
final volume with nuclease free H2O. First Strand Synthesis Master mix was added to
the sample mix and incubated at 42º C for 1 h, enzymes were heat inactivated 10 min at
70ºC, followed by a cooling to 4º C for 2 min. Subsequently, Second Strand Synthesis
Master mix was added to the sample mix, and incubated at 16ºC for 2 h. The reactions
were heat-inactivated by 10 min incubation at 75ºC.
The resulting cDNA was then
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processed in an IVT reaction to generate cRNA. The cRNA was used in first strand
synthesis reactions to generate target of the correct sense for hybridization to the
Affymetrix Gene and Exon arrays. The reactions were set up with 10g cRNA, random
hexamer, and first strand synthesis reagents to make cDNA. The targets were then
treated with Rnase to remove template RNA and cleaned up over kit supplied columns,
or Agencourt SPRI beads, following manufacturer’s protocol (RNAClean, #000494). A
total of 5.5g of the clean cDNA target was then enzymatically fragmented and endlabeled using the Affymetrix kit reagents. The cRNA, cDNA, and fragmented and endlabeled targets were assessed by Agilent bioanalysis to insure that the amplified targets
met the recommended smear range, and fragmentation and end-labeling were complete.
For both Exon and Gene arrays, the requisite amount of target was then added to
hybridization cocktail to give a final target concentration of ~25ng/l in the hybridization
cocktail. The targets in hybridization cocktail were heat denatured at 99ºC for 5 min,
cooled to 45ºC for 5 min, centrifuged and then loaded on the Affymetrix Hu Gene
1.0STcartridge arrays for a hybridization period of 16 h at 45ºC, with rotation of 60RPM.
The cartridge arrays were washed, stained and scanned per standard Affymetrix
Protocols using Affymetrix Hybridization Wash and Stain kit reagents (#900720).
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