Peg it virus precipitation solution

The protocol for lentivirus production and transduction have been described elsewhere.³⁵ Briefly, 293T cells were transfected with four µg of plasmid and four µg of the lentiviral vectors using Lipofectamine‐3000 according to the manufacturer's protocol (Invitrogen). PEG‐it virus precipitation solution (SBI System Biosciences) was added to the supernatant, and ultracentrifugation was performed to collect concentrated viral particles. Hepatocytes were transduced with lentiviral particles with 6 μg/ml polybrene (Invitrogen).

For construction of the recombinant lentiviruses that expresses specific shRNAs against MECOM or PU.1, the targeted sequences (see S2 Table) were synthesized and inserted into the pLentiLox 3.7-RNAi plasmid (Invitrogen) following the manufacturer’s protocols. For construction of the recombinant lentivirus that expresses miR-22, a 300-bp DNA fragment containing the miR-22 precursor was amplified and inserted into pMiRNA1 vector. The miRZip lentivector construct expressing miRZip shRNAs targeting miR-22 (Lenti-ZIP-miR-22) was purchased from SBI (Mountain View, CA, USA). The virus packaging was performed using a packaging kit from SBI (Mountain View) according to the manufacturer’s instructions. The lentivirus particles (Lenti-miR-22, Lenti-Con, Lenti-ZIP-miR-22, Lenti-ZIP-Con, shMECOM, shPU.1, shCon) were harvested and concentrated using PEG-it Virus Precipitation Solution (SBI). The lentiviral particles were added into the THP1 cells or CD34⁺ cells in the presence of Polybrene (5 μg/mL; Sigma, St. Louis, MO, USA). The cells were washed with PBS 24 hours after infection and exposed to lineage-specific differentiation cultures or plated for colony-forming assay.

TRIM8 cDNA was obtained from pLenti‐TRIM8‐mGFP TrueORF clone (Origene#RC205812L2; Origene, Rockville, MD, USA), with the vector control mGFP cDNA from pLenti‐C‐mGFP TrueORF clone (Origene#PS100071). The TRIM8 shRNA lentiviral plasmid in pGFP‐C‐shLenti vector was obtained from Origene (Martinez et al., 2006; TL300821). Lentivirus packaging plasmids psPAX2 (Addgene#12260; Addgene, Cambridge, MA, USA) and pMD2.G (Addgene#12259) were cotransfected with cDNA or shRNA into HEK293T cells with Lipofectamine 3000 transfection reagents (Martinez et al., 2006; L3000015). Supernatant was collected and concentrated by precipitation with PEG‐it virus precipitation solution (Cat#LV810A‐1; SBI, Palo Alto, CA, USA) according to the protocol. Viral pellets were resuspended in 1× PBS and kept at −80 °C for stock or directly used in cell transduction. Gene expression efficiency was confirmed by GFP expression under fluorescence microscopy and western blot analysis.