MCL cell lines Jeko-1, Granta-519 and Rec-1 were purchased from DSMZ (Braunschweig, Germany). Cell line MAVER-1 was developed in our laboratory [60
]. Cell line Mino was obtained from ATCC (Manassas, VA, USA). UPN-1 cells were kindly provided by Dr. Elias Campo (Barcelona, Spain). Due to over-sensitivity to DMSO-induced apoptosis, Rec-1 cells were not used for apoptosis-induction assays.
Tumor samples Tumor samples were collected from the archives of the Department of Pathology and Diagnostics of the University of Verona. Informed consent had been collected for all patients and procedures were carried out according to the ethical guidelines of the University Hospital of Verona with the approval of an ethics committee, in compliance with the Helsinky Declaration.
PhosphoScan analysis was performed as previously described [23
] on MCL cell lines MAVER-1, Granta-519, Jeko-1, and Rec-1.The phospho-proteins identified using this analysis were categorized into KEGG Pathways using the DAVID Ease web framework [61
]. For P-value and fold enrichment calculation the complete list of human genes was considered as a background. The calculation was repeated for lists obtained by selecting genes above the abundance cut-offs of 3, 4 and 5.Illustrations were produced using the R statistical software package (http://www.r-project.org
Flow cytometric analysis of several tyrosine-phosphorylated forms of Syk (Y323, Y352 and Y525) and downstream target phospho-BLNK was performed as previously described [38
] using antibodies shown in Table . All experiments were performed in triplicate.
Antibodies used in the study
RNA isolation and cDNA synthesis RNA isolation was performed using the Allprep DNA/RNA/Protein Mini Kit (Qiagen GmbH, Hilden, Germany), and RNA quality was assessed by using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and RNA 6000 Nano chips (Agilent Technologies). Each cDNA was synthesized from 1 μg total RNA using random primers and the Superscript III First-strand synthesis system (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.
The analysis of SYK
isoforms was performed as previously described [58
]. Reaction products were analyzed by the Agilent 2100 Bioanalyzer and the DNA1000 Chip (Agilent Technologies).
Quantitative RT-PCR mRNA expression analysis was performed on ABI PRISM 7900HT Fast Real-time PCR System (Applied Biosystems, Foster City, CA) using Power SYBR green PCR Master Mix (Applied Biosystems). Oligonucleotide primers used were: CCND1-F AACTACCTGGACCGCTTCCT, and CCND1-R GGGGATGGTCTCCTTCATCT; TBP-F, GCACAGGAGCCAAGAGTGAA, and TBP-R, TCACAGCTCCCCACCATATT.TATA box binding protein (RefSeq ID NM_003194.3) transcript level was used to normalize SYK
expression.Calibration curves for each couple of primers were obtained by serial dilution of cDNA. Expression data were analyzed by the comparative threshold cycle (Ct) method accordingly to User Bulletin No. 2 (Applied Biosystems). All experiments were performed in triplicate. The statistical significance of the data was investigated by Student’st
-test. All P values were two sided and considered significant when less than 0.05.
Syk inhibition experiments
Piceatannol (Sigma-Aldrich Chemie GmBH, Steinheim, Germany) was resuspendend in DMSO as a 100 mM stock. Cells were diluted at 500*103
/ml in RPMI 1640 (1% fetal calf serum) and let grow for 16 h. Piceatannol was then added at concentrations between 1 μM and 80 μM (depending on the sensitivity of cell lines). Cells were harvested at 24 and 48 h and apoptosis levels analyzed by Annexin V (Annexin V–FITC apoptosis detection kit I, BD) as previously described [11
]. All experiments were performed in triplicate.
Western blotting Immunoblotting was performed by standard methodology, using the antibodies and dilutions indicated Table . Protein content of samples was measured using a colorimetric method (DC protein assay, Bio-Rad). All lanes were loaded with the same amount of total protein (in duplicate or triplicate), and were also visually verified by Ponceau red staining after electroblotting.
Immunofluorescence Immunofluorescence was performed using the antibodies shown in Table (both primary and secondary antibodies were diluted 1:100). Briefly, cells were washed in PBS, deposited on charged slides by gravity, fixed in cold methanol (−20°C) for 20 min and then air-dried. Cells were then re-hydrated, incubated with protein blocking solution for 10′, then incubated serially with primary and secondary antibodies (with three washes in PBS in-between). Slides were examined under fluorescent light using an Olympus BX61 microscope. Images were acquired using appropriate filters via a monochromatic camera cooled CCD camera (iAi, Japan) and analyzed using the Olympus Cytovision software.