Rm igf 2

LSK cells were transduced overnight with control or gene-specific shRNAs and then cultured at 15,000 cells/well in non-tissue culture–treated 96-well plates for 5–6 d in serum-free expansion medium (STEMCELL Technologies) with 10 ng/ml RM SCF, 20 ng/ml RM Tpo, 20 ng/ml RM IGF-2 (PeproTech), 10 ng/ml RH FGF-1 (R&D Systems), and 10 µg/ml heparin (Sigma-Aldrich). Cells were collected 5–6 d after plating and stained with the following antibodies: (B220, CD3, CD4, CD8, CD19, Gr-1, and Ter119)-PerCP, Sca-1-PerCP-Cy5.5, and c-Kit-APC-780. After staining for surface antigens, cells were labeled with Annexin V-FITC (BD) and DAPI, and then analyzed using an LSR Fortessa and FlowJo version 9.4.11.

For analysis of CFU potential of LSK cells after knockdown of screen hits, LSK cells were transduced overnight with control or gene-specific shRNAs, and then cultured at 15,000 cells/well in non-tissue culture–treated 96-well plates for 5–6 d in serum-free expansion medium (STEMCELL Technologies) with 10 ng/ml RM SCF, 20 ng/ml RM Tpo, 20 ng/ml RM IGF-2 (PeproTech), 10 ng/ml RH FGF-1 (R&D Systems), and 10 µg/ml heparin (Sigma-Aldrich). 500 mCherry⁺ LSK cells were then isolated by FACS and plated in M3434 methylcellulose (STEMCELL Technologies). For CFU analysis of Foxa3^+/+ or Foxa3^−/− HSCs, 150 HSCs (LSK CD150⁺CD48⁻) were isolated by FACS from WBM, and then plated in M3434. Colonies were analyzed 10 d after plating.

LSK cells were transduced overnight with control or gene-specific shRNAs and then cultured at 15,000 cells/well in nontissue culture treated 96-well plates for 5–6 d in serum-free expansion medium (STEMCELL Technologies) with 10 ng/ml RM SCF, 20 ng/ml RM Tpo, 20 ng/ml RM IGF-2 (PeproTech), 10 ng/ml RH FGF-1 (R&D Systems), and 10 µg/ml heparin (Sigma-Aldrich). mCherry⁺ LSK cells were then collected by FACS and stained with the following antibodies: (B220, CD3, CD4, CD8, CD19, Gr-1, and Ter119)-PerCP, Sca-1-PerCP-Cy5.5, and c-Kit-APC-780. Cells were then fixed using the Cytofix/Cytoperm kit (BD), followed by staining for Ki67-FITC (Clone SolA15; eBioscience) and DAPI. Cells were analyzed via an LSR Fortessa and FlowJo version 9.4.11.