Murine scf and tpo

FACS-sorted LT-HSCs (100 cells per well) were cultured for 7 days in SFEM supplemented with 100 ng ml⁻¹ murine SCF and TPO, 20 ng ml⁻¹ murine IL3 and IL6 (Peprotech) and 5 U ml⁻¹ human EPO (PromoKine). Viable cells were assessed using the ViaLight Plus Cell Proliferation and Cytotoxicity BioAssay Kit (Lonza) at days 3, 5 and 7 according to the manufacturer's instructions. Luminescence was measured using a Mithras LB940 luminometer (Berthold Technologies). For ectopic miR-193b expression experiments, FACS-sorted LT-HSCs (100 cells per well in 96-well format) were lentivirally transduced (MOI=100) and cultured for up to 9 days in SFEM supplemented with 100 ng ml⁻¹ SCF and TPO. Viable cells were counted with Trypan blue exclusion. The percentage of transduced cells (eBFP⁺) was analysed via FACS (BD CantoII). Ectopic expression of the unrelated miRs-132/212 in LT-HSCs using the same lentiviral expression strategy served as an additional control. FACS-sorted GMPs (300 cells per well in 96-well format) were lentivirally transduced (MOI=20) and cultured for up to 7 days in SFEM supplemented with 100 ng ml⁻¹ SCF and 20 ng ml⁻¹ murine IL3 and IL6 (Peprotech). The percentage of transduced cells (eBFP⁺) was analysed via FACS.

FACS-sorted LT-HSCs (100 cells per well) from miR-193b^−/− and miR-193b^+/+ mice were cultured in SFEM (Stemcell Technologies) supplemented with 100 ng ml⁻¹ murine SCF and TPO, 20 ng ml⁻¹ murine IL3 and IL6 (Peprotech) and 5 U ml⁻¹ human EPO (PromoKine). Cells were analysed via FACS with antibodies against CD117 and CD16/32 and a dead/live cell exclusion (Fixable Viability Dye, 0.1 μl per test, eBioscience). During myelomonocytic differentiation, all CD117⁺ cells (CD117⁺ CD16/32⁻) first start to express CD16/32 (CD117⁺ CD16/32⁺) before they lose CD117 expression (CD117⁻ CD16/32⁺).