SUPPLEMENTARY METHODS
Rifat A. Hamoudi,1 Alex Appert,1 Hongtao Ye,1 Liping Gong,1 Ming-Qing Du1
1
Department
of
Pathology,
University
of
Cambridge,
Cambridge,
CB2
0QQ,
U.K.;
Gene expression microarray: Total RNA was isolated using the RNeasy kit (Qiagen, Valencia, CA)
and the RNA integrity was assessed using an Agilent 2100 Bioanalyzer. Double stranded cDNA was
generated from 2-5g of total RNA, followed by in vitro transcription with biotin-labeled nucleotides
using the Affymetrix RNA transcript labeling kit (Affymetrix Inc, Santa Clara, CA).
Biotinylated
cRNA was purified and hybridized to Affymetrix GeneChip HG-U133A (MALT lymphoma) or
Affymetrix H133 plus 2 (FL and MCL).
Except where indicated, transcriptome analyses were
performed using R system software packages (version 2.8.0) including Bioconductor (version 2.0) and
in house software written using R code. Quality control analysis of the microarray hybridization was
performed using Affymetrix GCOS software. A scale factor within mean plus 2 standard deviation
and percentage present calls >20% were used as threshold values and only those passed these criteria
were used for subsequent analyses according to the criteria recommended by the Tumor Analysis Best
Practices Working Group.1 All microarray data have been deposited with Gene Expression Omnibus
(GEO; http://www.ncbi.nlm.nih.gov/geo/, GSE18736).
Normalization: Raw gene expression data from Affymetrix CEL files were uploaded to bioconductor
where MAS5 and gcRMA normalization were performed separately for each Affymetrix platform.
MAS5 data were scaled to a target intensity of 100. The gcRMA normalized data were imported into
Genespring 7.3.1, log-transformed and median centered. For comparison between microaray data
obtained from HG-U133A and HG-U133 plus2, an additional median polishing normalization step
was applied. gcRMA normalized data were used for unsuprevised clustering analysis and gene set
enrichment analysis (GSEA), while MAS5 normalized data were used for identification of
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differentially expressed genes between MATL lymphoma with and without translocation as suggested
previously. 2 Both MAS5 and gcRMA normalized data were used in non-specific filtering.
Non-specific filtering: To filter out non-variant genes, a combination of noise and variance filtering
was applied. To filter out non-expressed genes, only probes with a value of 50 or higher in the MAS5
dataset in 2 or more samples were selected.
To eliminate non-variant genes, only those with a
coefficient of variation (CV) value of 10% or higher in the gcRMA dataset across all cases were
considered to be variant and thus selected. Finally, the genes that passed the above two filtering
methods were intersected to obtain a common set of variant genes.
For comparison of microarray data between HG-U133A and HG-U133 plus2 platform where
indicated, the non-specific filtering was similarly performed separately for each platform as above,
then intersected to generate a final common set of variant genes. All the above analyses were
performed using scripts written in R. The above procedure for analysis of expression microarray data
from HG-U133A and HG-U133 plus2 platform was validated by a serial empirical testing using the
published pulmonary MALT lymphoma expression microarray data from the HG-U133 plus2 platform
as a reference.3
Quantitative reverse-transcription polymerase chain reaction (qRT-PCR)
The expression of selected genes identified from the expression microarray analysis was validated by
qRT-PCR with 18SrRNA as an internal control.4 Briefly, total RNA was isolated from tumor cells
microdissected from paraffin-embedded tissue sections,5 and treated with Turbo DNAse. cDNA was
synthesized using gene specific primer (Supplementary Table S4) and qPCR was performed in
triplicate using an iCycler iQ system (Bio-Rad, Hertfordshire, UK) with SYBR Green-I .4
Immunohistochemistry: BCL10, MALT1, BCL2, CD69 and CD86 protein expression were
investigated by immunohistochemistry under the conditions detailed in Supplementary Table S5. The
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immunostaining was evaluated independently by two assessors (HY, MQD) and scored according to
the percentage of positivity (<30%, 30-70%, >70% cells) and staining intensity (strong, moderate,
weak, negative). Cases were considered positive when 30% tumor cells were stained.
Western blot analysis: Protein extracts from frozen tissues or cultured cells were mixed with SDS gel
loading buffer, denatured, separated on 4-12% Bis-Tris precast gradient gels using the NuPAGE
electrophoresis system (Invitrogen, Paisley, UK).
The proteins were electrotransferred onto an
Immobilon PVDF membrane, then incubated with a primary antibody, followed by appropriate HRPconjugated secondary antibody and detected using an Immobilon chemiluminescent HRP substrate
(Millipore, Watford, UK). The filter was stripped and similarly re-probed with antibody to -actin or
others.
Where indicated, Western blots were quantified using an Advanced Image Data Analyzer
(Version 4.18, Raytest, Straubenhardt, Germany).
NF-B reporter assay: The full length coding sequence of BCL10 and API2-MALT1 was cloned into
a modified pIRESpuro2 (Clontech, Saint-German-en-Laye, France) expression vector. The expression
constructs of TLR1, TLR2 and TLR6 in pFLAG-CMV1 vector were kindly provided by Dr. Kuwano.6
The capacity of these expression constructs to induce NF-B activation was measured using the Dual
Luciferase Reporter Assay System (Promega, Southampton, UK) in Jurkat T-cells. Briefly, a total of
9g DNA containing 2g of each expression vector, appropriate amount of vector control, 1.5g
pNFkB-luc (a Firefly luciferase reporter for NF-B activity), 1.5g pRL-TK (a Renilla luciferase
reporter as a control) was transfected into a total of 8106 Jurkat T-cells using Amaxa with solution T
and program T16 (Amaxa, Cologne, Germany). The transfected cells were dispatched into 6 to 8
wells of 24-well plates, left for 20 hours to recover and then treated with 10g/ml LPS or vehicle for 6
hours. The cells were harvested, washed with PBS, lysed and assayed for luciferase activities using a
VICTOR3 luminometer (PerkinElmer, Beaconsfield, UK) following the manufacture’s instructions.
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References
1 The Tumor Analysis Best Practices Working Group. Expression profiling--best practices for data generation
and interpretation in clinical trials. Nat Rev Genet 2004; 5:229-237.
2 Pepper SD, Saunders EK, Edwards LE, Wilson CL, Miller CJ. The utility of MAS5 expression summary and
detection call algorithms. BMC Bioinformatics 2007; 8:273.
3 Chng WJ, Remstein ED, Fonseca R, Bergsagel PL, Vrana JA, Kurtin PJ et al. Gene expression profiling of
pulmonary mucosa-associated lymphoid tissue lymphoma identifies new biologic insights with potential
diagnostic and therapeutic applications. Blood 2009; 113:635-645.
4 Ye H, Gong L, Liu H, Hamoudi RA, Shirali S, Ho L et al. MALT lymphoma with t(14;18)(q32;q21)/IGHMALT1 is characterized by strong cytoplasmic MALT1 and BCL10 expression. J Pathol 2005; 205:293-301.
5 Pan LX, Diss TC, Peng HZ, Isaacson PG. Clonality analysis of defined B-cell populations in archival tissue
sections using microdissection and the polymerase chain reaction. Histopathology 1994; 24:323-327.
6 Shimizu T, Kida Y, Kuwano K. A triacylated lipoprotein from Mycoplasma genitalium activates NF-kappaB
through Toll-like receptor 1 (TLR1) and TLR2. Infect Immun 2008; 76:3672-3678.
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