Mission shrna vectors

Lentiviruses were generated after subcloning the PITPNA cDNA sequence into the expression vector pCCL-cPPT-PGK-IRES-WPRE (Addgene). The resulting construct was transfected along with packaging plasmids pMD2.G and pSPAX2 (Addgene) into HEK293T cells (ATCC, CRL-3216). Cell culture media containing the virus was collected 48 and 72 h after transfection, concentrated, and stored at −80 °C. Knockdown of PITPNA by MISSION shRNA vectors (Sigma-Aldrich) was confirmed in human pancreatic 1.1B4 cells and isolated islets. Human islets were treated with non-overlapping shRNAs against the human PITPNA mRNA (accession number NM_006224), and TRCN00000299703 (SHCLNV 06302009MN) was used for all studies. TRC2 pLKO.5 Lentiviral Transduction Particles (pLKO.5-puro non-Mammalian shRNA Control Plasmid DNA; SHC00204V) were used to treat control human islets. Polybrene (Santa Cruz Biotechnology, Cat# sc-134220, Texas, USA) was added to the media with the final concentration of 10 μg/ml before infection. In brief, 250 islet equivalents (IEQ) seeded in each 12-well plate were infected with each lentivirus at an M.O.I of 20 for 48–72 h to ensure complete infection. All plasmids and shRNAs related to these procedures are listed in Supplementary Table 3.

Lentiviruses were produced by transfection in 293T with MISSION shRNA vectors (Sigma-Aldrich). CML CD34⁺ cells (4 × 10⁶ cells/ml) were infected by spinoculation (800 g, 90 min, 30 °C). Two adjuvants, poloxamer 407 (100 μg/mL, Sigma-Aldrich) and prostaglandin E2 (10 μM, Merck), were added to the culture medium [50 (link)]. Cells were harvested 72 h later, and the knockdown effect was examined by western blot analysis.

Diagnostic bone marrow or peripheral blood from AML patients and cord blood were collected with informed consent; patient clinical characteristics were outlined in Supplementary Methods. Normal human CD34⁺ cells were isolated as previously described [17 (link)].
Cell lines were obtained from ECACC^TM (Salisbury, UK) or ATCC (Middlesex, UK) and cultured under recommended conditions. The genetic identity of the cell lines was confirmed by short tandem repeat (STR). Cells at passages greater than twenty were not used in the experiments performed in this study. Monitoring for Mycoplasma contamination was performed using the MycoAlert Detection Kit (Sigma). S100A4 harboring a nuclear localization sequence (NLS) was expressed utilizing retroviral and lentiviral vectors co-expressing GFP as a selectable marker (Supplementary Methods). For knock down studies, Mission® shRNA vectors based on TRC(1)2-pLKO.5-puro (S100A4 shRNA and nonmammalian shRNA control) were purchased from Sigma-Aldrich, Dorset, U.K. CD34⁺ cells and cell lines were transduced and cultured as previously described [17 (link), 18 (link)].

Mission shRNA vectors purchased from Sigma were transiently transfected along with pCMV-VSVG and ps-PAX2 into HEK 293T (ATCC #CRL-3216) cells using polyethylenimine (PEI) at a ratio of 3 µL of 10 µg/µL PEI per 1 µg of plasmid (shRNA information provided in Supplementary Table 5b). HEK 293T cells are validated and authenticated by short tandem repeat (STR) analysis and undergo mycoplasma testing on a yearly basis. Viral supernatant was collected 48- and 72 h post-transfection and filtered through a 0.45 µm syringe filter. Viral supernatant was added to NRAS-null MEFs along with 10 µg/mL polybrene. Fresh media was placed on the cells the following day and 1.5 µg/mL puromycin selection began 48 h post-infection.