mTORC1 and mTORC2 during development of huMCs from CD34⁺ progenitors, in neoplastic MC lines, and in cells with D816V-mutated KIT from patients with systemic mastocytosis. (A) Peripheral blood CD34⁺ progenitors were cultured for 8 weeks. (B) mTORC1 and mTORC2 were then analyzed by immunoblotting and compared with total cell numbers (dashed lines) during the course of 8 weeks. (C-D) mTORC1 and mTORC2 were analyzed in huMCs, LAD2, HMC-1.1, and HMC-1.2 MCs by immunoblotting. (E) Expression of mTOR, raptor, and rictor mRNA in normal cells (healthy volunteers, n = 10) or cells with the KIT D816V mutation (n = 13) of donor's BM mononuclear cell fraction aspirates was analyzed, and differences in mRNA expression of each protein between the group of normal and KIT D816V donors and (F) correlation of mRNA expression of the mTORC components within each group of donors evaluated. In panels B and D, data represent means (cell counts) or means and SEM (phosphorylation and protein levels; n = 3; 3 donors or 3 independent sample preparations). In panel B, differences between phosphorylation or protein expression in 0- and 1-week-old cells, respectively, and 0- and 8-week-old cells are indicated (*P < .05 by t test). In panel D, differences between huMCs and each type of the myeloproliferative MCs are indicated (*P < .05 by t test). In panel E, bars represent mean of values determined in each group and differences between the group of normal and KIT D816V donors are indicated (*P < .05 by t test). In panel F, the correlation was evaluated by Spearman correlation test between normal donors (n = 10) and KIT D816V mutation donors (n = 13).