Whole Genome Amplification (WGA) of genomic DNA was performed with REPLI-g Mini/Midi Kit (QIAGEN, Hilden, Germany). Briefly, 10 ng of genomic DNA was denatured by adding denaturation buffer. After denaturation has been stopped by addition of neutralization buffer, a master mix containing buffer and DNA polymerase is added. The isothermal amplification reaction proceeded overnight at 30°C.
One µl of WGA product was amplified by PCR in a 25 µl solution containing PCR Buffer 10X (20 nM tris-HCl, 50 nM KCl), 1.5 nM MgCl2, 5 nM dNTPs, 2.5 U Taq DNA polymerase (Roche, Basel, Switzerland) and 10 pmol of each primer. Polymerase Chain Reaction (PCR) reactions were composed by: one activation cycle of 95°C for 10', 35 cycles of a denaturing step of 95°C for 30'', a specific annealing step for each couple of primers, and an elongation step of 72°C for 30'', followed by a final elongation step of 72°C for 7'. Primers and specific annealing conditions for PCR amplification of all PRDM1 coding exons and TP53 (exons from 4 to 8) are described in Table 3. The PCR products were purified using QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany). Purified amplicons were sequenced directly from both strands (Mycrosynth, Switzerland) and compared with corresponding germ-line sequences (NM_001198.3, BLIMP1;
NM_000546.5, TP53) with BLAST software. All mutations were confirmed on independent PCR products, and germ-line polymorphisms, including changes listed in the NCBI SNP database or present in available matched normal DNA, were excluded.
Identity of matched normal DNA was verified by analyzing known polymorphisms.
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Table 3. Primers for PRDM1 and TP53 sequencing.
GENE Primer FW Primer REV annealing
PRDM1-ex1 5'-TGACGCCAAACACATGTTAAA-3' 5'-GTTCCAGCTCACACTCGTCA-3' 60°C 30 sec PRDM1-ex2 5'-TATACGGCTTCTTGGCTCTT-3' 5'-AGGAACAGTTGAAGGCTGG-3' 58°C 1 min PRDM1-ex3 5'-AGATGGTCTCCCCCTATGGT-3' 5'-AAGCAAGCAACAAACTGTTTC-3' 58°C 1 min PRDM1-ex4 5'-GCCCTGATTTCTGCTGATTC-3' 5'-GTCCCTAGCTTAAGCCACCT-3' 60°C 45 sec PRDM1-ex1b 5'-TAGATGTTCATCCCGTTCTGA-3' 5'-ACTTGAGAATGACCAAAATG-3' 56°C 1 min 30 sec
PRDM1-ex5 5'-TTGAGTGAGTGGCCCAGAG-3' 5'-AGGGAAGTCACTTGTCCAAA-3' 60°C 1 min 30 sec PRDM1-ex6 5'-AAACTCCCTGCTAGCCTGTG-3' 5'-GCCATCTCAAGTCATCAGCA-3' 60°C 45 sec PRDM1-ex7 5'-CACAAGGAGGCTTCTCACCT-3' 5'-GATTTCAGTAACTTTGGAGTT-3' 58°C 1 min
TP53-ex4 5'-TCCTCTGACTGCTCTTTTCAC-3' 5'-TGAAGTCTCATGGAAGCCAG-3' 60°C 30 sec TP53-ex5 5'- GTTTCTTTGCTGCCGTCTTC-3' 5'-AGCAATCAGTGAGGAATCAG-3' 58°C 30 sec TP53-ex6 5'-TCTGATTCCTCACTGATTGCTC-3' 5'-CCACTGACAACCACCCTTAAC-3' 61°C 30 sec TP53-ex7 5'-TCATCTTGGGCCTGTGTTATC-3' 5'-AGTGTGCAGGGTGGCAAG-3' 60°C 30 sec TP53-ex8 5'-AGGACCTGATTTCCTTACTGCC-3' 5'-ATAACTGCACCCTTGGTCTCC-3' 60°C 30 sec
Methylation
The methylation status of BLIMP1 was investigated by bisulfite sequencing PCR method.
500 ng of DNA used as template was chemically modified by bisulfite treatment (EZ DNA Methylation Kit, ZYMO Research, Irvine, CA, USA). This technique involves treating methylated DNA with bisulfite, which converts unmethylated cytosines into uracil.
Methylated cytosins remain unchanged during the treatment. Once converted, the methylation profile of DNA can be determined by PCR amplification followed by DNA sequencing. PCR amplification was performed with primers specific for converted bisulfite converted DNA, designed with Methprimer tool. 2 µl of bisulfite converted DNA was amplified by PCR in a 25 µl solution containing PCR Buffer 10X (20 nM Tris-HCl, 50 nM KCl), 1.5 nM MgCl2, 5 nM dNTPs, 2.5 U Taq DNA Polymerase (Roche, Basel, Switzerland) and 30 pmol of each primer. The thermal profile consisted in: one activation cycle of 95°C for 10', 35 cycles of a denaturing step of 95°C for 30'', an annealing step of 58°C for 30'', and an elongation step of 72°C for 30'', followed by a final elongation step of 72°C for 7'. PCR products were separated by agarose gel electrophoresis and visualized with Alphimager 3400 (Alphainnotech Corporation, Fremont, CA, USA). Negative control (DNA not treated) was used in each reaction. The PCR products were purified using
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QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany). Purified amplicons were sequenced directly from both strands (Mycrosynth, Switzerland) and analyzed for the methylation detection.
2. H UMAN C ELL C ULTURE Cell Lines
We used different established human cell lines derived from Anaplastic Large Cell Lymphoma (ALCL): SUDHL1, Supm2, TS, JB6, L82, Karpas 299 (ALK+ ALCL) and FE-PD, MAC-1 (cALCL, commonly used as ALK- ALCL model). All of them were cultured with RPMI 1640 media supplemented with fetal bovine serum (10%) (PAA, Pasching, Austria) and Penicillin-Streptomycin-Neomycin (~5,000 units penicillin, 5 mg streptomycin and 10 mg neomycin/mL, Sigma-Aldrich, St Louis, MO, USA).
HEK-293T packaging cells (ATCC) were cultured in DMEM (GIBCO) medium complemented with 10% of FBS (PAA) and Penicillin-Streptomycin-Neomycin (~5,000 units penicillin, 5 mg streptomycin and 10 mg neomycin/mL, Sigma-Aldrich, St Louis, MO, USA). All the cell lines have undergone karyotypes analysis (conventional or with the addition of FISH probes when needed, done by our collaborator M.G. Tibiletti) and search for Mycoplasma infection by biochemical assay (Lonza AG, Cologne, Germany). Controls have been repeated during the course of the project.
Transfection
Lipofectamine is a common trasfection reagent (Invitrogen, Life technologies, Zug, Switzerland) used to introduce siRNA or plasmid DNA into in vitro cell cultures by lipofection. Lipofectamine reagent contains lipid subunits that can form liposomes in an aquaeous environment, which capture the transfection materials, such as DNA plasmids.
The DNA-containing liposomes can fuse with the plasma membrane of living cells, create pores along the membrane allowing nucleic acids to cross into the cytoplasm and contents to be available to the cell for replication or expression. For each condition, 0.4 × 106 or 0.8 × 106 cells were plated in a 24-wells plate in 500 µl of medium. Two different mixes were prepared: the first one containing 2 µl of lipofectamine in 50 µl of Optimem medium (GIBCO, Life technologies, Zug, Switzerland) and the second one containing 1 µg of DNA plasmids in 50 µl of Optimem medium. After having incubated both mixes for
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5' at room temperature, we combined them and incubated the final mix for 20' at room temperature to allow the formation of complexes between DNA and lipofectamine. Then, we added the 100 µl DNA-lipofectamine complexes directly to each well containing cells and mixed gently by rocking the plate back and forth. Cells were incubated for 4 hours at 37°C in a CO2 incubator. We tried different conditions of transfection, removing and changing the medium or not. At different time point, cells were controlled for the expression of GFP with a fluorescence microscope.
Electroporation
Transfection of ALCL cell lines was carried out also by electroporation using a Nucleofector device (Amaxa, Lonza AG, Cologne, Germany). For each condition, 5 × 106 cells were resuspended in 82 µl of Nucleofector solution V (Nucleofector kit) together with 18 µl of supplements and 2 µg of plasmid used for control (pWPI) or plasmid for the re-expression of BLIMP1 (pWPI-HA-BLIMP1) (kindly provided by Pasqualucci L.), both of them containing green fluorescent protein (GFP). Cells were then placed in an electroporation cuvette. Transfection with each plasmid was carried out with the application of different electroporation programs, G-016 and A-030. Immediately after electroporation, 500 µl of prewarmed RPMI medium with 10% FBS was added to the cuvette and cells were transferred into culture plates containing prewarmed medium. At different time point, cells were controlled for the expression of GFP with a fluorescence microscope.