Diap3 (Dia2) knock-down in murine marrow-derived mesenchymal stem cells

Control (non-targeting) and Dia2-targeting siRNA were transfected into murine-derived mesenchymal stem cells and mRNA-Seq analysis was performed two days after transfection. Murine marrow‐derived mesenchymal stem cells (mdMSC) were isolated from murine marrow and maintained in IMDM as described (Peister et al., 2004). Cells (passage 6-12) were seeded into 6-well plates at a density of 6,000 cells/cm2 in IMDM and allowed to adhere for 24 hours. Cells were then transfected overnight with control or Dia2 siRNA (50 nM, 5′-CAGAGTCCATGATTCAGAACTTAAT) by Lipofectamine RNAiMax (Invitrogen) and Opti-MEM. Next morning, transfection media was replaced with αMEM. RNA was harvested twenty four hours later (RNeasy mini kit, Qiagen). Peister, A. et al. Adult stem cells from bone marrow (MSCs) isolated from different strains of inbred mice vary in surface epitopes, rates of proliferation, and differentiation potential. Blood 103, 1662-1668 (2004).