Expression of miR-130a and Smad4 during granulopoiesis. (A) Cytospins of purified cells displaying the structure of the 4 subpopulations isolated from BM (MBs and PMs, MCs and MMs, and BCs and SCs), and from peripheral blood (PMN). The arrow indicates cells of increasing maturity. Images were acquired on a Olympus BX51 (60×/1.40 PlanApopchromat oil objective, florescences free immersion oil (Applichem) microscope using Olympus DP 70 camera and analyzed with analySIS B software package (Olympus). (B) The average miRNA expression levels in 6 donors determined by real-time PCR. The SD refers to differences between donors. (C) The relative expression level of Smad4 mRNA in the isolated subpopulations determined by real-time PCR after normalization to β-actin. Data are shown as the mean ± SD from triplicate measurements. (D left) Representative immunoblot of Smad4 protein expression in the 4 isolated subpopulations. The right panel shows the relative expression of Smad4 normalized to β-actin expression. The experiment was repeated 3 times with similar results. The highest value among those being compared was assigned the value 1, and the relative expression of the other samples was recalculated accordingly.