Isolation of Mononuclear Cells (Mncs) and CD34+ Cells from Bone Marrow Or Peripheral Blood

SupplementaRY information: MaterialS AND Methods

Isolation of Mononuclear Cells (MNCs) and CD34+ Cells From Bone Marrow or Peripheral Blood Samples. For in vitro experiments, Bone Marrow (BM) or Peripheral Blood (PB) Mononuclear Cells (MNCs) were isolated by Ficoll-Paque (GE Healthcare, Waukesha, WI, USA) density-gradient centrifugation, according to the manufacturer’s protocol. CD34+ cells were obtained from total MNCs after immunomagnetic separation using the CD34 Micro Beads kit (Miltenyi Biotec, Bergisch Gladbach, Germany) following the manufacturer’s instructions. All analyses were performed on samples from patients at baseline, and subsequently before each cycle of azacitidine administration.

Antibodies and reagents. The following antibodies and reagents were purchased from commercial sources. PI-PLCbeta1 mouse primary antibody was from Millipore/Upstate (Billerica, MA, USA, #05-164), whilst mouse Cyclin D3 (#2936) primary antibody was from Cell Signaling Technology (Beverly, MA, USA). For immunocytochemical analyses, the secondary antibodies were fluorescein isothiocyanate (FITC)-conjugated F(ab’)2 fragment of sheep anti-mouse IgG (#2883, Sigma-Aldrich, St Louis, MO, USA). For Chromatin Immunoprecipitation (ChIP) experiments, antibodies against Methyl-CpG Binding Domain Proteins (MECP2, sc-101017; MBD1, sc-10751; MBD2, sc-10752; MBD3, sc-166319; and MBD4, sc-10753), as well as anti-Sp1 (sc-59X) anti-CEBPa (sc-036-61X), anti-c-myb (sc-036-74512X) and anti-MZF-1 (sc-036-66991X) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Analysis of Gene and Protein Expression. The amount of PI-PLCbeta1a (Hs01008373_m1, Applied Biosystems) and PI-PLCbeta1b (Hs01001939_m1, Applied Biosystems) splicing variants, as well as Cyclin D3 mRNA (Hs00236949_m1, Applied Biosystems) were assessed by applying a semi-quantitative Real-Time PCR method (1, 2). For all genes tested, the GAPDH gene (Hs99999905_m1, Applied Biosystems) was considered as the internal reference and data were statistically analyzed by the GraphPad Prism software (v.3.0, La Jolla, CA, USA), using healthy subjects as the calibrator. As for protein analysis, immunocytochemical experiments were performed on freshly isolated BMMNCs or PBMNCs, which were collected by centrifugation at a density of 0.3x106 cells/mL and immunostaining analysis was performed as previously described (3). For quantification of immunoreactivity, at least 50 to 100 cells/slide were counted.

Chromatin Immunoprecipitation (ChIP) analysis. Chromatin extracted from MDS samples at baseline and during azacitidine therapy was sonicated into fragments approximately 500bp in size and immunoprecipitation was performed with specific primary antibodies, against Methyl-CpG Binding Domain Proteins (MECP2, MBD1, MBD2, MBD3 and MBD4) and the four selected transcription factors (Sp1, CEBPa, c-myb and MZF-1). RNA Polimerase II antibody or normal IgG were used as positive and negative controls. The precipitated DNA fractions were then amplified by a Sybr-Green-based semi-quantitative Real-Time PCR method using specific primers for PI-PLCbeta1 promoter sequence (forward: 5′- CTCCAGCTAGAGCCAAAAGC-3′; reverse: 5′- ACTTTGCACGCTGGGTCAG-3′) and for GAPDH, used as a housekeeping gene (forward: 5′-CAATTCCCCATCTCAGTCGT-3′; reverse: 5′-TAGTAGCCGGGCCCTACTTT-3′). PCR products were subsequently analyzed on a 1% agarose gel stained with Sybr-Green (Invitrogen Life Technologies, Carlsbad, CA, USA).

REFERENCES

1. Follo MY, Bosi C, Finelli C, Fiume R, Faenza I, Ramazzotti G, et al. Real-time PCR as a tool for quantitative analysis of PI-PLCbeta1 gene expression in myelodysplastic syndrome. Int J Mol Med 2006 Aug; 18(2): 267-271.

2. Follo MY, Finelli C, Mongiorgi S, Clissa C, Chiarini F, Ramazzotti G, et al. Synergistic induction of PI-PLCbeta1 signaling by azacitidine and valproic acid in high-risk myelodysplastic syndromes. Leukemia 2011 Feb; 25(2): 271-280.

3. Follo MY, Mongiorgi S, Bosi C, Cappellini A, Finelli C, Chiarini F, et al. The Akt/mammalian target of rapamycin signal transduction pathway is activated in high-risk myelodysplastic syndromes and influences cell survival and proliferation. Cancer Res 2007 May 1; 67(9): 4287-4294.