SUPPLEMENTARY INFORMATION: MATERIALS AND METHODS
Induction of Erythroid Differentiation in Normal Human CD34+ Cells and CD34+ Transfection.
Expansion of human normal CD34+ progenitor cells was obtained by following the first step of a three-step
published protocol (1), with minor modifications, as previously reported (2). Briefly, cell proliferation was
induced in a serum-free medium (Iscove’s Modified Dulbecco’s Medium, IMDM) supplemented with 4mM
L-glutamine, 50 U/ml penicillin, 50μg/ml streptomycin, 1% BSA, 120ug/ml human transferrin, 900 ng/ml
ferrous sulfate, 90 ng/ml ferric nitrate and 10μg/ml human recombinant insulin, 10−6 M-hydrocortisone, 100
ng/ml stem cell factor (SCF), 5 ng/ml interleukin-3 and 1 IU erythropoietin. The concentration of cells
ranged from 2×104 to 1.4×106 cells/ml. To induce erythroid differentiation, we followed a different published
protocol (3). Briefly, cells were harvested from the proliferation medium, washed with IMDM and cultured
at 1.8×105 to 5×105 cells/ml in IMDM in the presence of 20% FCS, 1U/ml erythropoietin and 10 ng/ml
human recombinant insulin. Viable cells were then counted with a hemocytometer and scored by Trypan
blue exclusion dye.
The full-length cDNA for human PI-PLCbeta1a and PI-PLCbeta1b (4) were cloned into pcDNA3.1 vector
(Life Technologies, Carlsbad, CA, USA) expression vector plasmid. Transient transfection of these vectors
into normal CD34+ cells was performed using an Amaxa Nucleofector™ apparatus (Lonza, Basel,
Switzerland), according to the manufacturer's instructions.
Normal CD34+ cells were cultivated in proliferation medium for 8 days. Then, cells were cultivated in
differentiation medium for 4 days or maintained in proliferation medium for 4 more days, for a total of 12
days. At the first day of differentiation medium or the ninth day of proliferation medium, cells were
transfected with 1 μg each of PI-PLCbeta1a and PI-PLCbeta1b vector, or 2 μg empty pcDNA vector. After 4
days of differentiation or 12 days of proliferation, cells were harvested and prepared for RNA extraction or
immunocytochemical experiments.
Antibodies and reagents. The following antibodies and reagents were purchased from commercial sources.
Mouse PI-PLCbeta1 (#05-164) and mouse PI-PLCgamma1 (#05-163) primary antibodies were from
Millipore/Upstate (Billerica, MA, USA), whereas FITC-conjugated EPO receptor antibody (FAB307F) was
from R&D Systems (Minneapolis, MN USA). On the other hand, mouse Cyclin D3 (#2936), rabbit Akt
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(#9272) and rabbit p-Akt Ser473 (#4058) primary antibodies were from Cell Signaling Technology (Beverly,
MA, USA). For immunocytochemical analyses, the secondary antibodies were fluorescein isothiocyanate
(FITC)-conjugated F(ab’)2 fragment of goat anti-rabbit IgG (#F1262, Sigma-Aldrich, St Louis, MO, USA)
and FITC-conjugated F(ab’)2 fragment of sheep anti-mouse IgG (#2883, Sigma Aldrich).
Gene Expression Analysis. The expression of several genes belonging to the nuclear inositide signalling
was quantified by using a specific TaqMan Real-Time PCR method (Applied Biosystems, Foster City, CA,
USA), as described elsewhere. In particular, the expression of PI-PLCbeta1a (Hs01008373_m1) and PIPLCbeta1b (Hs01001939_m1) splicing variants, Cyclin D3 (Hs00236949_m1), PI-PLCgamma1
(Hs00234046_m1), Beta-Globin (Hs00758889_s1), Gamma-Globin (Hs00361131_g1), EPO Receptor
(Hs00959427_m1), MAP2K3 (Hs00177127_m1) and MAP3K11 (Hs00176759_m1) were quantified. The
GAPDH gene (Hs99999905_m1) was used as the internal reference, as described elsewhere (5). Data were
statistically analyzed by the GraphPad Prism software (v.3.0, La Jolla, CA, USA), using healthy subjects as
the calibrator.
Protein Analysis. Freshly isolated bone marrow mononuclear cells were collected by centrifugation at a
density of 0.3x106 cells/mL and immunostaining analysis was performed as previously described (6). For
quantification of immunoreactivity, at least 50 to 100 cells/slide were counted. For quantification of EPO
receptor expression, we applied a flow cytometry approach, as described elsewhere (7).
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