S nitrosocysteine

Peripheral blood mononuclear cells were isolated from buffy coats of normal donors using lymphocyte separation medium (BioWhittaker, Walkersville, MD, USA). We performed a two-phase liquid culture protocol for erythroid differentiation, as previously described [13 (link)]. Briefly, after incubation in phase I culture, CD34+ cells were purified by negative selection using the StemSep cell separation method (Stem Cell Technologies, Vancouver, CA, USA). The CD34+ cells were resuspended in the phase II medium, which contained a mixture of cytokines including human recombinant erythropoietin (Amgen, Thousand Oaks, CA, USA). Erythroid cells were treated at different time points in the phase II medium, with S-nitrosocysteine (CysNO) and hydroxyurea (Sigma-Aldrich, St. Louis, MI, USA), incubated at 37 °C in a humidified atmosphere with 5% CO₂ [13 (link)]. The viable cell counts were performed by a trypan-blue exclusion technique (BioWhittaker). For isolation of total RNA from erythroid cells we used the RNeasy Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. Quantitative real-time PCR assay of γ- and β-globin mRNA transcripts was carried out with the use of gene-specific double-fluorescently labeled probes in a 7700 Sequence Detector (Applied Biosystems, Foster City, CA, USA) as previously described [13 (link)].