All experimental protocols were reviewed and approved by the University of British Columbia (UBC) Research Ethics Board (REB) (Certificate #H06-60016).
Cell Lines, Cell Culture Conditions
The human HL-derived cell lines KM-H2, L-428 and the ALCL cell lines DEL and SR-786 were obtained from the Deutsche ammlung von Mikrooganism und Zellkulturen GmbH (Braunschweig, Germany). All cell lines were cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (Cansera Inc., Etiobicoke, ON, Canada), L-glutamine and penicillin and streptomycin in a humid environment of 5% CO2 at 37°C to a density of 106 cells/ml.
Cell Lysate Preparation
HD lymphoma cell line, L-428, was cultured in IMDM complete media (10% FBS, with penicillin/streptomycin/fungizone, Gibco) for 48 hrs. The cultured cell was harvested and pelleted at ~1000 g for 5 minutes. The cells were washed in PBS (pH7.2) for 3 times and lysed in Mammalian protein extraction buffer supplied by Pierce. The samples were incubated on ice for 10 minutes and pelleted at 10,000 × g for 10 min. The supernatant was transferred to fresh tubes.
Formation of the Antibodies and Protein G Beads Complex
100 μl of protein G beads (50%) (Pierce) were added to each micro-centrifuge tube. The beads were pelleted at <2000 g for 30 seconds and washed in 1 ml of PBS once. 200 μl of PBS and 5 μg of antibodies were added to each tube. The beads were incubated at room temperature for at least 2 hrs and washed in PBS twice. Normal mouse IgG was coupled to the protein A beads in the same way and these beads are used for pre-clearing the lysates.
400 μl of the sample was incubated with normal mouse IgG-protein-A beads for 1 hr at room temperature and transferred into the tube containing mAb-protein A complexes. The beads were then incubated at 4°C overnight and washed in 1 ml of PBS for 3 times. The beads were resuspended in 1 ml of PBS and 50 μl of beads were transferred into another 1.5 ml tube. The beads were pelleted and as much buffer as possible was removed. The original tube was stored at -20°C.
SDS-Page and Western-Blot Analysis
25 μl of the 1 × SDS sample buffer was added to the second tube and heated at 100°C for 5 min. The tube was centrifuged 13000 ×g briefly and the sample was loaded to 4-20% Tris-Glycine gel (Bio-Rad). The samples were separated at 120 V for 80 min. The gels were equilibrated in pre-chilled 1× transfer buffer and the proteins were then transblotted onto a nitrocellulose membrane at 90 volts for 60 min. Western-blot analysis with R24.1/R23.1 was performed by blocking the membrane in 5% non-fat-dry milk, and probing with primary antibody (R24.1/R23.1) and secondary antibody (goat-anti-mouse IgG-AP) sequentially. The protein band which reacted with the antibody was visualized in BCIP/NBT substrate. The original tube was submitted to Kendrick Laboratories, Inc. (Madison, WI, USA) for 2D-gel analysis after the Western-blot analysis confirmed the candidate protein was pulled down by the antibody.
Surface Protein Labelling
Surface protein labelling with biotin was performed with the Pierce Cell Surface Protein Isolation Kit as per the manufacturer's instructions (Thermo Fisher Scientific, Fisher Canada, Nepean, Ontario). Briefly, Jurkat and L-428 cells were cultured in RPMI 1640 (STEMCELL Technologies, Vancouver, British Columbia, Canada) with 10% FBS, and 1× Antibiotic Antimycotic (GIBCO Invitrogen Canada Inc. Burlington, Ontario) for 48 hrs. Cells were harvested and washed twice in ice-cold PBS and counted. 2.5 × 107 cells were used for surface labelling. One vial of Sulfo-NHS-SS-Biotin was dissolved in 48 ml of ice-cold PBS. 24 ml of the biotin solution were added to each labelling tube. PBS was used to replace the Sulfo-NHS-SS-Biotin for the control tubes. The tubes were placed on a rocking platform for 30 minutes at 4°C. 500 μl of Quenching Solution were added to each tube to quench the reaction. 20 ml of TBS were added to each tube and the cells were centrifuged at 500 × g for 3 minutes. The cells were washed twice in 20 ml of TBS and lyzed in 500 μl of lysis buffer with protease inhibitor. The samples were transferred into 1.5 ml microcentrifuge tubes and incubated on ice for 30 min. The samples were vortexed every 5 min for 5 seconds and centrifuged at 10,000 g for 2 min at 4°C. 500 μl of the NeutrAvidin Agarose slurry were added to the spin column and washed with 400 ul of Wash Buffer for 3 times. The supernatants were incubated in the columns for 60 min at room temperature on with end-over-end mixing using a rotator. The columns were centrifuged at 1,100 × g for 1 min and the flow-throughs were transferred to fresh tubes. The columns were washed in 500 μl Wash buffer 4 times. 400 μl of SDS-Page sample buffer were added to each column and the columns were incubated at room temperature for 1 hr with the end-over-end mixing. The columns were centrifuged at 1,000 × g for 3 minutes and the eluted samples were analyzed on 4-20% Tris-HCl gel (Bio-Rad Laboratories (Canada) Ltd. Mississauga, Ontario). SDS-PAGE and Western-blot analysis was performed following the same protocol as described above.
2D-Gel Analysis and Coomassie-Blue Staining
Two-dimensional electrophoresis was performed according to the carrier ampholine methods  as follows: Isoelectric focusing was carried out in glass tubes of inner diameter 2.0 mm using 2% pH 3.5-10 (GE Healthcare, Piscataway, NJ) for 9600 volt-hrs. One μg of an IEF internal standard, tropomyosin, was added to each sample. This protein migrates as a doublet with lower polypeptide spot of MW 33,000 and PI 5.2; an arrow on the stained gels marks its position. The enclosed tube gel pH gradient plot for this set of ampholines was determined with a surface pH electrode. After equilibration for 10 min in Buffer 'O' (10% glycerol, 50 mM dithiothreitol, 2.3% SDS and 0.0625 M Tris, pH 6.8) the tube gels were sealed to the top of stacking gels on top of 10% acrylamide slab gels (0.75 mm thick) and SDS slab gel electrophoresis carried out for about 4 hrs at 12.5 mA/gel. The following proteins (Sigma Chemical Co., St. Louis, MO) were added as molecular standards to a well in the agarose that sealed the tube gel to the slab gel: myosine (220,000), phosphorylase A(94,000), catalyse (60, 000), actin (43, 000), carbonic anhydrase (29,000) and lysozyme (14, 000). These standards appear as bands at the basic edge of the Coomassie blue-stained 10% acrylamide slab gel. The gel was dried between sheets of cellophane with the acid end to the left.
After slab gel electrophoresis, the gel for blotting was transferred to transfer buffer (12.5 mM Tris, pH8.8, 86 mM Glycine, 10% MeOH) and transblotted onto PVDF paper overnight at 200 mA at approximately 100 volts/two gels. Gel ID numbers are indicated in the lower acid corner of the membrane.
Western-blot analysis was performed on PVDF paper. The PVDF paper was incubated in 5% non-fat dry milk for 2 hrs followed by an incubation in primary antibody (1:1000 diluted R24.1/R23.1) at 4°C overnight. The PVDF was washed in PBS 4 times and then incubated in goat-anti-mouse IgG-HRP conjugate (1:2000) at room temperature for 2 hrs. The paper was washed 4 times in PBS and incubated in 10 ml of SuperSignal West Pico Chemiluminescent substrate (Pierce Biotech., Rockford, IL) fro 10 min. A Kodak BioMAx MS film was exposed to the PVDF paper in a film cassette. The protein spot on a duplicate dried 2D gel was cut and submitted to Protein Chemistry Core Facility in Columbia University (New York, New York) for LC/MS/MS analysis.
RT-PCR and Sequence Analysis
cDNAs of CYB5B were amplified from mRNA with Clontech's Titanium One Step RT-PCR kit (Clontech, Mountain View, CA, USA). The forward primer is 5'-CTCAAGGAAAGTAGTCGCGG-3', which starts at the first nucleotide of 5' end of the mRNA. The reverse primer is 5'-AACTCTTGGCCTCAAGCGA-3', which starts at the nucleotide 660. A product of 660 bp is expected to be amplified using this primer pair. The amplified RT-PCR products were gel purified with Spinprep Gel DNA kit (Novagen, Gibbstown, NJ, USA). The purified samples were submitted to MacrogenUSA Inc. (Rockville, MD, USA) for sequencing.
The protein arrays used in this study are the PEX arrays from Imagenes (Berlin). Each array contains 16,000 Escherichia coli His-tagged expression clones from a variety of expression libraries arrayed in a 3 × 3 pattern on PVDF membranes. The expression vector used is pQE30NST (GenBank Accession No. AF074376) and it is transformed into E. coli strain SCS1 (Stratagene) [43, 44]. Sequence information for individual clones recognized by antibodies was provided by Imagenes.
Prior to antibody screening, the arrays are prepared by removing the dried colonies from the PVDF membranes with tissue paper and TBSTT (TBS supplemented with 0.05% Tween 20 and 0.5% TritonX-100). This is followed by three washes, for 20 min each, in TBST (TBS supplemented with 0.05% Tween 20) and blocked with 3% (w/v) milk powder in TBST for 3 h at room temperature, the filters were incubated overnight with the monoclonal antibody R23.1  at a 1:1000 dilution in 2% BSA/TBST. Primary antibody incubation was followed by three washes of 20 min each in TBST, and the protein arrays were then incubated with the secondary antibody, alkaline phosphatase-conjugated (AP) anti-mouse IgG (A1418; Sigma,) for 1.5 h. Following three washes for 20 min each in TBST, the filters were incubated for 10 min in AP buffer (1 mM MgCl2, 100 mM Tris-Cl, pH 9.5) and 5 min in 0.125 mM Attophos in AP buffer. Filters were illuminated with long-wave UV light (460 nm EPI) and images were taken using a high resolution CCD camera (Fuji LAS 3000; Fuji Film Global)). Image analysis was performed using VisualGrid (VisualGrid™ is available as a free download from (GPC Biotech, Munich, Germany) and the identities of detected clones were confirmed by sequencing.
Recombinant Protein Expression and Purification
The E. coli expression clones 9028C1242 and 9028A0834 from the PEX library were grown in 5 ml liquid cultures and protein expression induced with 1 mM IPTG and purified as previously described using nickel affinity chromatography [43, 45]. The size of each purified protein was determined using SDS PAGE and Coomassie staining.
The purified proteins were separated by SDS PAGE and transferred onto PVDF membranes at 100 V for 1 1/2 hr. The membrane was blocked for 2 hr in 3% Marvel/TBST, washed in TBS (3 × 10 min) and incubated for 2 hr with the primary antibody as described above. Antibody binding was detected using an alkaline phosphatase-conjugated anti-mouse, or anti-rabbit, IgG secondary antibody (Sigma) and visualized on a Fuji LAS 3000 imager.
SMRT-Array CGH and Gene Expression Profiling
Tiling path array comparative genomic hybridization was performed as described previously . Genomic DNA was extracted via standard proteinase K/RNAse treatment and phenol-chloroform extraction.
Briefly, 200 ng of test and reference DNA were separately labelled with Cyanine3-dCTP and Cyanine5-dCTP (Perkin Elmer Life Sciences Inc., Boston, MA, USA), respectively, via random priming at 37°C for 16-18 h in the dark. Labelled sample and reference DNA probes were combined, precipitated, re-suspended, blocked and hybridized onto BAC arrays containing 26,819 clones spotted in duplicate on a single slide. After hybridization, arrays were washed, rinsed, and dried using an oil free air stream. Slides were scanned using a charge-coupled device (CCD) based imaging system (arrayWoRx eAuto, Applied Precision, Issaquah, WA, USA). Images were analyzed using SoftWoRx Tracker Spot Analysis software (Applied Precision). The ratios were normalized using a stepwise normalization technique to remove any biases added from non-biological sources.
Custom software (SeeGH)  was used to visualize normalized data as log2 ratio plots. Genomic alterations were classified as homozygous deletions, loss, normal, gain, and amplification for ratios < -1.0, -1.0 to -0.15, -0.15 to 0.15, 0.15 to 1.0, and >1.0 respectively . SeeGH is now freely available from http://www.flintbox.ca/technology.asp?page=312.
Relative Expression of CYB5B in Cell Lines Using qRT-PCR and Affymetrix GeneChip® Human Genome U133 Plus 2.0 Arrays
Validation of the expression of CYB5B
was carried out by quantitative real-time PCR (qRT-PCR) using SYBR green chemistry. Total RNA from DB, OCI-LY19, KMH2, L428, DEL and SR-786 cell lines was extracted using TRIzol reagents (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. cDNA synthesis was conducted using SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen, Carlsbad, CA, USA) from the RNA samples and UHRR (Universal Human Reference RNA). GAPDH
was used as endogenous control. After first strand synthesis, an equivalent of 16 ng of cDNA were added to three triplicate PCR reactions containing SYBR GreenER qPCR SuperMix for ABI PRISM®
(Invitrogen, Carlsbad, CA, USA), 10 μM forward primer and 10 μM reverse primer in a final volume of 10 μl. The SYBR GreenER qPCR SuperMix contains ROX (6-carboxy-X-rhodamine), the passive reference fluorochrome that normalizes for pipetting volume errors. Each reaction was performed in triplicate in 384-well Optical reaction plates that cycled for 2 min at 50°C, 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds, and 60°C for 1 minute on 7900 HT Fast Real-Time PCR System (Applied Biosystems, CA, USA). Fluorescent data were converted into cycle threshold (CT) measurements using the 7900 HT-system sequence detection systems (SDS) software and were exported to Microsoft Excel. The Comparative CT Method calculates relative gene expression using the equation:
Affymetrix GeneChip® Human Genome U133 Plus 2.0 Arrays containing the CYB5B probe set 238554_at were used to assess the expression of CYB5B in HL cell lines in comparison to microdissected normal germinal centre B cells. Microdissection was preformed on a Zeiss Axioplan 2 microscope equipped with mmi CellCut Plus (Molecular Machines Industries'; Haslett, MI, USA) Technology using 6 μm sections from reactive tonsil tissue. Total RNA was extracted using Allprep extraction kits (Qiagen, ON, Canada) and cRNA preparation was performed following the routine protocol for 2-cycle labeling reactions. 11 μg of labeled cRNA were then hybridized on the array overnight and the arrays were washed, stained and scanned using Affymetrix Fluidics Station 450 and Affymetrix GeneChip Scanner (Affymetrix Inc.; Santa Clara, CA). All microarrays passed homogeneous criteria for testing quality control including present call rates >30% and normalized unscaled standard errors <1.05 (NUSE). Affymetrix gene expression data was pre-processed and normalized by Robust Multichip Average (RMA) in R using Bioconductor .
Flow Cytometric Analysis of Surface Expression of CYB5B on Bone Marrow CD34+ Cells
Bone marrow aspirates were depleted of red blood cells by either treating them with Red Blood Cell Lysis Buffer, or using by Ficoll-Paque Plus density gradient centrifugation (GE Healthcare); each method gave similar results. Treated cells were blocked with normal mouse IgG (Invitrogen) for 20 minutes at 4°C, then washed with 4 volumes PBS+2%FBS. Cells were stained using APC-conjugated anti-CD34 (1/50, StemCell Technologies) and FITC-conjugated R23.1 antibody (1/50), for 45 minutes at 4°C. Cells were again washed with PBS+2%FBS, then resuspended in PBS+1 ug/mL propidium iodide, incubated on ice 10 minutes, then fixed with an equal volume of 2% formaldehyde. Cells were then analyzed on a BD FacsCalibur and using FCS Express software.
RT-PCR Analysis of CYB5B Expression in Normal Peripheral Blood and Bone Marrow Samples Enriched for T and B Cell Subpopulations
Cells were separated using Easy-Sep CD3+ or CD19+ magnetic bead-conjugated antibodies (Stem Cell Inc.). RNA was extracted using Trizol reagent (Invitrogen) and up to 2.5 ug of total RNA was reverse-transcribed (Superscript II, Invitrogen). GAPDH primers were used to normalize cDNA loading; when appropriate, cDNAs were diluted using the same buffer composition as used to perform reverse transcription. GAPDH and CYB5B PCRs were carried out in duplicate tubes under the same cycling conditions and run out on the same gel at the same time (1× TAE, 2% agarose). Bands were quantified using Fluorchem 3.04A (Alpha Innotech Corporation).
PCR conditions: 95°C 1 min, 94°C 15 s-56°C 15 s-72°C 15 s for 32 cycles, 72°C for 5 minutes, hold at 4°C.
Primers (designed using Primer3, http://frodo.wi.mit.edu/) were as follows:
hGAPDH-R: TTGATGGTACATGACAAGGTGCGG (160 bp)
hCYB5B-R: GGATTTGCTTTCCGATGTGTA (197 bp)
Flow Cytometric Analysis of Surface and Cytoplasmic CYB5B Protein Expression in Cell Lines
Jurkat T-cells (5 × 106 per tube) were blocked with 1/50 control mouse IgG, then labelled on the cell surface with 1/50 of R23.1-FITC, then washed twice with PBS+2%FBS, stained for non-viable cells using propidium iodide, and fixed in 1% formaldehyde. Alternatively, isotype control IgG-blocked cells were resuspended in Cytofix/Cytoperm solution (BD Biosciences Cytofix/Cytoperm Fixation/Permeabilization Solution Kit), then incubated with 1/100 of R23.1-FITC conjugated mAb. Cells were analyzed using a BD FACSCalibur and FCS Express (version 3, DeNovo Software, Los Angeles).
For blocking experiments, Jurkat T-cells (5 × 106 per tube) were blocked with 1/50 isotype control mouse IgG1, then washed and resuspended in Cytofix/Cytoperm solution as per manufacturer's directions. Permeabilized cells were then resuspended in 25 uL and incubated with isotype control IgG1 or unlabelled R23.1 antibody (1/25 of either) for 30 minutes at 4°C. Then, 25 uL containing 0.5 uL R23.1-FITC was added, and cells were incubated an additional 30 minutes at 4°C. Cells were analyzed using a BD FACSCalibur and FCS Express (version 3, DeNovo Software, Los Angeles).