SUPPLEMENTARY MATERIALS & METHODS
mRNA expression and real-time PCR
Total RNA was isolated from fresh tissues by lysis in TRIzol reagent (Invitrogen). B
cells were purified by negative selection with a mouse B-cell isolation kit (Miltenyi Biotec), and
total RNA was isolated with an RNeasy Mini kit (QIAGEN). RNA was reverse-transcribed (RT)
to cDNA with a RETROscript kit (Ambion). An ABI PRISM 7900HT sequence detection
system (Applied Biosystems) was employed for quantitative real-time PCR. The primer/probe
sets for murine bcl-2 and GAPDH were from TaqMan Assays-on-Demand Products (Applied
Biosystems). The TaqMan bcl-2 primer/probe set detects total bcl-2 transcripts. Bcl-2 gene
expression levels were normalized to GAPDH.
Western blot
Cells were lysed in cell extraction buffer (Biosource) with addition of protease inhibitor
cocktail set III (Calbiochem). Equal amounts of protein from each sample were separated on a
13% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane (Amersham
Biosciences), and probed with antibody to Bcl-2 (sc-7382, Santa Cruz Biotechnology) or -Actin
(A2130, Sigma). Immunocomplexes were detected by incubation with horseradish peroxidaseconjugated secondary antibody and the ECL detection system (Amersham Biosciences).
Viability assay
Purified B lymphocytes from spleens were cultured in RPMI 1640 containing 10% FBS,
1 mM L-glutamine, and antibiotics. Cells were seeded in 96-well microplates at a density of 2.5
× 106/ml. Viability was determined using the MTT Cell Growth Determination kit (Sigma
Aldrich, St. Louis MO) according the manufacturer’s instructions. The MTT kit is a colorimetric
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assay that measures mitochondrial activity by the cleavage of 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyl tetrazolium bromide (MTT). Cells were assayed every 24 hours up to 3 days.
Cell cycle analysis
Single-cell suspensions were washed with cold phosphate-buffed saline (PBS), fixed in
cold 70% ethanol overnight, and washed again with cold PBS, 5mM EDTA. Cells were stained
with propidium iodide-RNase A solution (BD Pharmingen) with incubation at 37°C for 1 hour in
the dark, and analyzed using a FACs Caliber flow cytometer (Becton Dickinson) and Cell Quest
software. A total of 10,000 events were collected using the FL2 channel for PI intensity and
relative DNA content. Forward scatter and side scatter gates and doublet discrimination plots
were used to capture whole and individual cell cells respectively. Markers were used to quantify
the percentage of cells distributed in phases of the cell cycle.
Tissue
Lymph nodes and spleens from IgH-3’E-bcl2 (n=20; 7 to 14 months old) and WT (n=3; 8
to 13 months old) mice were fixed in formalin, embedded in paraffin, sectioned, and stained with
hematoxylin and eosin (H&E). Pieces of lymph nodes and spleens were also frozen in OCT
compound for immunohistology staining (n=6 knock-in and 3 WT mice). Mesenteric lymph
node with germinal centers from an adult C57Bl/6 mouse was frozen in OCT compound as a
positive control for the immunohistology staining.
Immunohistology staining
Semi-serial frozen sections were cut, fixed in acetone, and stained for germinal center B
cell markers that are expressed by human follicular lymphoma (CD19, CD45R/B220, BCL6, and
binding of peanut agglutinin (PNA))(1-3); for markers of infiltrating T cells (CD4 and CD8) (4)
and follicular dendritic cells (5) ; and with negative control antibodies as follows:
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Rat mAb. Frozen sections were sequentially incubated with a purified rat mAb to CD4
(clone GK1.5; 1:100 dilution in phosphate buffered saline (PBS)), CD8 (53-6.7; 1:100), CD19
(1D3; 1:100), CD45R/B220 (RA3-6B2; 1:200) or follicular dendritic cells (FDC-M1; 1:100)
(Pharmingen, San Diego, CA), or with an isotype-matched negative control mAb (Pharmingen)
for 1 hour at room temperature; second and third stage antibodies (per manufacturer’s
instructions; Rat on Mouse HRP-polymer Kit; Biocare Medical, Concord, CA), and Betazoid
DAB solution (per manufacturer’s instructions; Biocare Medical). There were three washes in
PBS after each incubation step. Slides were counterstained with hematoxylin.
BCL6. Frozen sections were sequentially incubated with rabbit polyclonal IgG antibody
to N-terminal BCL6 ((N-3): sc-858, Santa Cruz Biotechnology, Santa Cruz, CA; 1:200 in PBS)
or with normal rabbit IgG; biotinylated-anti-rabbit IgG (Vector Laboratories, Burlingame, CA;
1:400 in PBS with 2% normal mouse serum); peroxidase-streptavidin (Jackson ImmunoResearch
Laboratories, West Grove, PA; 1:400 in Tris buffer), and Betazoid DAB solution. There were
three washes in PBS after each incubation step. To improve visualization of BCL6 nuclear
staining, the slides were not stained with hematoxylin.
PNA. Frozen sections were sequentially incubated with biotinylated-PNA (Vector
Laboratories; 1:400 in PBS); peroxidase-streptavidin (1:400 in Tris buffer); and Betazoid DAB
solution. There were three washes in PBS after each incubation step. Slides were counterstained
with hematoxylin.
Evaluation of histology and immunohistology stains
H&E-stained paraffin sections were evaluated by light microscopy by a hematopathologist
(S.M.) without knowledge of the mouse age or strain. Lymphomas were diagnosed using criteria of
the WHO classification (6).
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Immunoperoxidase-stained frozen sections were evaluated by light microscopy by a
hematopathologist (S.M.) for the location of the DAB (brown) staining on the slides stained with
PNA or specific-antibody, as compared to the appropriate negative controls (7).
Photomicroscopy
Slides were photographed with a Nikon MICROPHOT-FXA microscope (Melville, NY)
with plan apochromat objectives (4x/0.10 NA, 10x/0.25, 20x/0.40 and 40x/0.65), a SPOT Insight
Color Mosaic camera (model 14.2; Diagnostic Instruments, Sterling Heights, MI), and SPOT
Advanced imaging software (version 4.6). Composite figures were produced with Adobe
Photoshop CS5 (version 12.0.1) and Adobe Indesign CS5 (version 7.0.2) (Adobe Systems, San
Jose, CA, USA). Some images were processed to improve color balance and brightness;
processing was applied equally across the entire image.
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Rodig SJ, Shahsafaei A, Li B, Dorfman DM. The CD45 isoform B220 identifies
select subsets of human B cells and B-cell lymphoproliferative disorders. Hum
Pathol 2005 Jan; 36(1): 51-57.
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Rose ML, Habeshaw JA, Kennedy R, Sloane J, Wiltshaw E, Davies AJ. Binding of
peanut lectin to germinal-centre cells: a marker for B-cell subsets of follicular
lymphoma? Br J Cancer 1981 Jul; 44(1): 68-74.
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Dvoretsky P, Wood GS, Levy R, Warnke RA. T-lymphocyte subsets in follicular
lymphomas compared with those in non-neoplastic lymph nodes and tonsils. Hum
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Scoazec JY, Berger F, Magaud JP, Brochier J, Coiffier B, Bryon PA. The dendritic
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cell types: an immunohistochemical study of 48 cases. Hum Pathol 1989 Feb; 20(2):
124-131.
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Harris NL, Jaffe ES, Diebold J, Flandrin G, Muller-Hermelink HK, Vardiman J, et
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1419-1432.
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Xu B, Wagner N, Pham LN, Magno V, Shan Z, Butcher EC, et al. Lymphocyte
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