Sonic Hedgehog Produced by Bone Marrow-Derived Mesenchymal Stromal Cells Supports Cell Survival in Myelodysplastic Syndrome

Figure 3

Modulation of Shh signaling influences the survival of MUTZ-1. MUTZ-1 cells were cultured in the presence of medium, exogenous Shh peptide (Shh-N) (500 ng/mL), cyclopamine (Cyc) (20 μM), and Shh-N + cyclopamine for 48 h, and cell survival was determined by the CCK8 and Annexin V-PI assays. There was significantly increased proliferation of MUTZ-1 cells in the presence of exogenous Shh peptide and decreased cell proliferation in the presence of cyclopamine, as determined by the CCK8 assay (a). Cyclopamine at concentrations of 10, 20, and 40 μM induced inhibition of cell proliferation ((b), ×200) and increased apoptosis (c) in MUTZ-1 cells within 48 h in a dose-dependent manner. Expression of GLI1 protein in CD34⁺ stem cells from healthy donors, MUTZ-1 cells, and SKM-1 cells shown by confocal laser scanning microscopy (d). Real-time PCR analysis of Smo, Gli1, and DNMT1 after treatment with Shh-N (500 ng/mL) and/or cyclopamine (20 μM) for 72 h (e). Expression of beta-actin (ACTB) was used as a housekeeping control gene. Fold-change was calculated with method compared with controls. Results are expressed as average ± SEM of triplicates each. .

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