SUPPLEMENTARY MATERIALS AND METHODS
Tissue samples
Normal tissues: Fresh tonsil was obtained from patients attending the Ear, Nose and Throat
Department, Radcliffe Infirmary, Oxford. Paraffin-embedded normal tissues were obtained
from the Departments of Histopathology and Paediatric Pathology of the John Radcliffe
Hospital, Oxford and the Centro Nacional de Investigaciones Oncologicas (CNIO), Madrid.
Normal peripheral blood cells: Peripheral blood samples were obtained from healthy
volunteers and used to make blood smears. Buffy coats from normal donors were obtained
from the National Blood Service, Bristol, UK. Mononuclear cells were separated by density
centrifugation using Histopaque 1077, (Sigma-Aldrich Co. Ltd., Dorset, UK) and some of the
cells were used to make cytocentrifuge preparations. CD20-positive peripheral blood B cells
were also purified by magnetic cell separation using the MidiMACs system (Miltenyi Biotec
Ltd., Surrey, U.K.) and cultured for 44 hours with and without Staphylococcus aureus Cowan
strain 1 (1:20,000 wt/vol) and recombinant IL-2 (5 ng/ml, Sigma-Aldrich Co. Ltd) before
cytocentrifuge preparations were made.
Neoplastic tissues: Tissue microarrays (TMAs) of hematopoietic tumors were obtained from
CNIO. TMAs of 107 cases of previously untreated DLBCL were obtained from the British
Columbia Cancer Agency (RDG). Scores of zero and one (0,1)(<30% of the cells positive)
were considered to be negative while scores of two and three (2,3)(>30% of the cells labeled)
were considered to be BCL11AXL-positive. Kaplan-Meier estimates of progression free
survival and overall survival were determined and the univariate and multivariate significance
of differences in actuarial survival evaluated by the log-rank and Cox regression methods
employing SPSS.
All normal and neoplastic cells and tissues were used only after informed consent had been
obtained.
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Cell lines
The KM-H2 (HL) and the Granta 519 (mantle cell lymphoma) lines were obtained from the
German Collection of Micro-organisms and Cell culture, Braunschweig. The DLBCL-derived
cell lines SUDHL-6, SUDHL-10, DB, OCI-Ly3 and OCI-Ly10 lines were provided by Drs E.
Davis and A. Rosenwald, Bethesda. The derivation of all other cell lines used in this study has
previously been reported. All cell lines were cultured in RPMI 1640 medium containing 10%
fetal calf serum (Gibco Life Technologies, Paisley) at 37oC in 5% CO2. Cells were used to
prepare cytocentrifuge preparations, cell pellets or fixed in 10% formol-saline for paraffin
embedding.
Generation of immunogen
A XhoI fragment spanning zinc fingers 4-6 (aas 637 – 835) of BCL11AXL was cloned into the
SalI site of the GST bacterial expression vector, GEX-KG. The resulting pGEX-KGBCL11A456 cDNA was transformed into Escherichia coli BL21, and protein expression
induced by adding 2.5 mM isopropyl-D-galactosidase (0.1 mM final concentration). After
2 hours, the cells were sonicated in phosphate buffered saline (PBS) and 1% Triton X-100
(Sigma Aldrich Chemical Co., U.K.) and following centrifugation, the supernatant was mixed
with glutathione-agarose beads and incubated for 1 hour at 4oC with agitation. The GSTBCL11A456 recombinant protein, comprising the COOH-terminus of BCL11AXL was eluted
with 50 mM Tris-Cl containing 15 mM reduced glutathione and used for subsequent
immunisation and screening purposes. This GST-BCL11AXL protein contained the entire
unique C-terminal region present in the XL isoform and 105 aa common to the C-terminus of
the L isoform.
Production of transfectants
The constructs pCR3.1-HABCL11AS, pCR3.1-HABCL11AL and pCR3.1-HABCL11XL
containing cDNA encoding HA-tagged short, long and extra-long BCL11A isoforms were
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prepared as described by Hiu Liu et al. (manuscript in preparation). The BCL11B construct
pIRES BCL11Bv was kindly provided by Dr G.K. Przybylski, Polish Academy of Sciences,
Poznan, Poland.
COS-1 cells at 80-90% confluency were transfected with one of the following constructs,
pCR3.1-HABCL11AS, pCR3.1-HABCL11AL, pCR3.1-HABCL11XL, pIRES BCL11Bv or vector
only using the Fugene 6 transfection reagent following manufacturer’s instructions (Roche
Diagnostics, Indianapolis, USA). After 3 hours culture the proteasome inhibitor N-Acetyl-Leu
Norleu A1 (ALLNL) (20 mM stock solution in DMSO, Sigma Aldrich Company Ltd.,
Gillingham, Dorset, U.K.) was added to give a final concentration of 100 M. The cells were
incubated for a further 20 hours and cytocentrifuge preparations made.
Production of antibodies
Balb/c mice were injected subcutaneously with 100 g of the BCL11AXL recombinant protein
emulsified in Titremax adjuvant (Stratech Scientific Ltd., Soham. U.K.). Two further
immunizations of 100 g recombinant protein in PBS were given at intervals of 10 days.
Three days after a fourth injection, spleen cells were fused with the NS1 myeloma cell line for
hybridoma production. Initial screening was carried out using an ELISA technique against the
recombinant BCL11AXL-GST protein and GST alone and immunolabelling of BCL11A
transfectants. Cloning by limiting dilutions was performed to produce a monoclonal antibody.
Source of antibodies
Monoclonal antibodies: Antibodies to CD8 (C8/144), CD31 (By126), CD32 (KB61), CD45
(2B11 and PD7/26), CD68 (KP1), CD74 (By2), CD79a (JCB117), CD163 and anti-leucocytespecific protein-1 (LSP-1) were produced in the LRF Immunodiagnostics Unit. Antibodies to
CD4 (T4-10), CD20 (DAKO-L26), CD34 (Q-Bend-10), CD68 (PGM1), vimentin (V9),
cytokeratin (LP34), myeloperoxidase (MPO7) and -actin were obtained from
DakoCytomation while anti-CD123 was obtained from (TCS, Biosciences Ltd., Botolph
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Claydon, UK). Three commercially available mouse monoclonal antibodies to murine
Ctip1/Bcl11a, raised against epitopes within aa regions 1-171 (15E3AC11), 172-434 (14B5)
and 434-776 (18B12DE6), were obtained from Abcam (Cambridge, UK).
Polyclonal antibodies: Anti-CD3 (DAKO-CD3 – diluted 1:100), anti-mouse Ig-conjugated to
horseradish-peroxidase (HRP) diluted 1:100 and the Envision staining kit were obtained from
DakoCytomation. Rabbit antibodies raised against either the zinc finger domains or the Cterminus of murine Bcl11b (antibodies Bcl11b/z and Bcl11b/c, respectively) were the kind
gift of Prof. Ryo Kominami, Okinawa, Japan. Goat anti-rabbit and -mouse isotype specific
antibodies conjugated to fluorescein-isothiocyanate (FITC) (diluted 1:100) were obtained
from Molecular Probes (Cambridge, U.K.).
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