MCL samples and cell lines
Peripheral blood mononuclear cells were obtained from 14 MCL leukemic patients by Ficoll-Hypaque density gradient (Stem Cell Technologies, Grenoble, France). Lymphocytosis was greater than 8.0×109/L and 10 out of 14 samples contained at least 80% of B lymphocytes (Additional file 5: Table S1). All B lymphocytes are monoclonal tumor B cells as evidenced through flow cytometry phenotyping of the surface immunoglobulin light chain (monotypic kappa or lambda). Seven cases (7/14) showed mutated IGHV and none of them displayed mutation in ITAM sequences of CD79B. The diagnosis of MCL was ascertained by immunophenotyping, cytogenetic and FISH analysis of t(11;14) and overexpression of cyclin D1 was detected by competitive RT-PCR according to the World Health Organization classification. All patients were homogeneously treated in a prospective trial of the GOELAMS group “Lymphome du manteau 2006 SA” . All patients were provided written informed consent, validated by the Ethics Committee from the GOELAMS group, in accordance with the Declaration of Helsinki. Patients usually received treatment very quickly after sampling, making it difficulties to repeat all experiments several times on the same sample. Jeko-1, and Granta-519 cell lines were purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunshwieig, Germany) and the HBL-2 cell line was a generous gift from Dr B. Sola (Caen, France) .
Patients’ cells were either used freshly isolated or cryopreserved in liquid nitrogen in the presence of 10% dimethyl sulfoxide and 20% heat-inactivated FCS. MCL leukemic cells (3 × 106 cells/ml) were cultured in complete RPMI 1640 medium supplemented with 10% heat-inactivated foetal calf serum. Jeko-1, HBL-2 and Granta-519 cell lines were maintained in culture in the same media. For BCR stimulation, plates were pre-coated with rabbit anti-human IgM antibody (10 μg/mL; Jackson ImmunoResearch, Baltimore, MD) as previously described  or the anti-IgM antibody was added to the culture medium at the same concentration for short stimulation time.
Antibodies and reagents
Antibodies to EGR-1 (mAb 44D5), c-MYC (mAb 9B11), phospho-Src family (mAb 100 F9) also reactive with phospho-Tyr397 LYN (catalytic site) and phospho-JNK (Thr183/Tyr185) were from Cell Signaling (Beverley, MA). Monoclonal mouse antibody (H-6) and polyclonal rabbit antibody (44) to LYN were from Santa-Cruz (Santa Cruz, CA). Anti-phosphotyrosine monoclonal antibody (clone 4 G10) was from Millipore (Billerica, MA). Dasatinib (Clini Sciences, Montrouge, France) was used at 100nM, unless otherwise stated. JNK inhibitor SP600125 and PP2 (4-Amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolo[3,4-d]pyrimidine) was from Sigma (Saint Quentin Fallavier, France) and (5Z)-7-Oxozeaenol (TAK1 inhibitor) was from Tocris Bioscience (Bristol, UK).
RT2 profiler PCR arrays
Tumor B-lymphocytes from MCL patients were purified by the RosetteSep® Human B Cell Enrichment Cocktail (Stemcell technologies, Grenoble, France). Cells were cultured for 3 hours upon BCR stimulation or left untreated. Total RNA were extracted and analyzed with “p53 signaling pathway” array (SA Biosciences PAHS-027) according to the manufacturer’s instructions (SA Biosciences, Frederick, MD) with an Applied Biosystems 7500 Fast Real-Time PCR Systems. Each gene expression was normalized to the mean Ct values from the four housekeeping genes available in the PCR array (β2-microglobulin, β-actin ribosomal protein L13A, hypoxanthine phosphorybosyl transferase-1 and β-actin), then normalized to unstimulated control cells to determine the fold-change. Relative fold change of expression was calculated by the ΔΔCt method and the values are expressed as 2-ΔΔCt. All points were done in duplicate.
Cell apoptosis was evaluated using flow cytometry (FACSCantoTM II Becton Dickinson) on leukemic MCL PBMC after gating on CD19+ cells using Annexin V-FITC and propidium iodide staining (BD Biosciences, San Jose, CA). Percentage of apoptotic cells corresponded to% of annexin V-positive, including PI-negative and PI-positive cells. All measurements were done in duplicate and the mean is indicated.
Quantification of EGR-1 and c-MYC mRNA by qRT-PCR
RNA from unstimulated or anti-IgM stimulated cells were extracted using RNeasy Mini kit (QIAGEN) and EGR-1 and c-MYC expressions were analyzed by qRT-PCR using SYBR Green reagents. Results were normalized to the mean Ct values from cyclophilin A housekeeping gene then normalized to unstimulated control cells to determine the fold change. Relative fold change of expression was calculated by the ΔΔCt method and the values are expressed as 2 -ΔΔCt. All points were done in duplicate. The primers used for amplification (Sigma-Aldrich) were as follows:
EGR-1 forward primer (5′CGAGCAGCCCTACGAGCACCTGAC3′),
EGR-1 reverse primer (5′TGCGCAGCTCAGGGGTGGGCTCTG3′),
c-MYC forward primer (5′GCTGCTTAGACGCTGGATTTTT3′),
c-MYC reverse primer (5′ACCGAGTCGTAGTCGAGGTCAT3′),
cyclophilin A forward primer (5′GCACTGGAGAGAAAGGATTTGG3′) and
cyclophilin A reverse primer (5′AGTGCCATTATGGCGTGTGA3′).
Western blotting and immunoprecipitation
Total protein extracts from 3×106 MCL cells (PBMC) were separated on 10% polyacrylamide denaturing gel, transferred to a nitrocellulose membrane and incubated overnight with the appropriate antibody followed by a secondary horseradish peroxidase-conjugated antibody (Bio-Rad). Detection was performed using ECL (Amersham Biosciences, Buckinghamshire, UK) and autoradiography (Biomax, Kodak, Rochester, USA). For LYN immunoprecipitation, HBL-2 cells were lysed in 1% Nonidet P-40 (NP-40) lysis buffer for 30 minutes on ice. Insoluble material was removed by centrifugation at 27 000 g for 10 minutes at 4°C and soluble proteins were immunoprecipitated with a rabbit anti-LYN (H-6) antibody for 2 hours at 4°C. Immunocomplexes were solubilized in SDS sample buffer, analyzed on SDS-PAGE, transferred and subjected to immunoblotting as described above using either a mouse anti-phosphotyrosine antibody (clone 4 G10) or a mouse anti-LYN (44) antibody.
Three million cells were resuspended in 100 μL of Human B Cell Lymphoma Nucleofector® Kit (Amaxa) containing either 1 μM of EGR-1 siRNA (On-Target plus SMART pool Human EGR-1, Dharmacon) or 1 μM of control siRNA (Ctrl1: On-Target plus non-targeting pool; Ctrl2: On-target plus non-targeting siRNA#1; Dharmacon). Cells were transfected in a Nucleofector II device (Lonza) by using U-015 program, transferred to culture plates and western blot and apoptosis assays were performed as described above.
Differences between groups were determined using the Student’s t test. Statistical analyses were performed using GraphPad Prism software (San Diego, CA). P values below 0.05 were considered statistically significant.