For knockdown of IL1RAP, we cloned shRNA template oligonucleotides into the pSIH1-H1-copGFP shRNA vector (System Biosciences) and produced lentiviral particles as previously described (Barreyro et al., 2012 (link)). THP-1, HEL, or TF-1 cells were transduced with a nonsilencing control (luciferase) shRNA- or IL1RAP-specific shRNAs (MOI = 1), and transduced (GFP+) cells were FACS sorted before transplantation. For all shRNA transduction experiments, 8 µg/ml polybrene was added to cells before incubation with virus, and spin infection was performed after addition of virus (1,000 rcf for 1 h at 32°C) to facilitate transduction. Knockdown efficiency was measured by flow cytometry using anti–human IL-1RAcP/IL-1R3 APC or PE (FAB676A and FAB676P; R&D) and mouse IgG1 APC (17-4714; eBioscience) or PE (IC002P; R&D) as controls.
Primary human MDS and AML MNCs were transduced with IL1RAP shRNA lentiviruses (MOI = 1). 24 h after shRNA infection, GFP+ cells were FACS sorted and plated between 1.5 × 105 and 2.5 × 105 cells/ml in human complete methylcellulose medium (HSC003; R&D) supplemented with 40 µg/ml low-density lipoproteins (L7914; Sigma-Aldrich). Colonies were scored after 10–14 d.
Primary human MDS and AML MNCs were transduced with IL1RAP shRNA lentiviruses (MOI = 1). 24 h after shRNA infection, GFP+ cells were FACS sorted and plated between 1.5 × 105 and 2.5 × 105 cells/ml in human complete methylcellulose medium (HSC003; R&D) supplemented with 40 µg/ml low-density lipoproteins (L7914; Sigma-Aldrich). Colonies were scored after 10–14 d.