Lentiviral Knockdown of ASM in CD4+ T Cells

Healthy CD4⁺ T cells were infected separately with an empty shRNA vector control (sh-C, pLKO.1-puro) or different human ASM shRNA (sh-1: NM_000543.2-367s1c1, TRCN0000049014; sh-2: NM_000543.2-1467s1c1, TRCN0000049015) lentiviral transduction particles (Invitrogen), according to the manufacturer's instructions. Recombinant lentiviral particles were produced by transient transfection of 293FT cells as described.^{40 (link)} CD4⁺ T cells were infected with lentiviral particles according to previously described method with a minor modification.^{41 (link)} Briefly, after stimulation with anti-CD3/28 antibodies (both 2 μg/ml) and human recombinant IL-2 (R&D Systems) (1 ng/ml) for 24 h, CD4⁺ T cells were infected by lentiviral particles by centrifugation at 2300 r.p.m. for 60 min at room temperature in the presence of polybrene (8 μg/ml). These cells were replaced with fresh media containing human recombinant IL-2 (R&D Systems) (1 ng/ml) for additional 48 h, and then selected with puromycin (2.25 μg/ml) for 5–10 days. The surviving cells were selected using a Dead Cell Removal Kit (Miltenyi Biotec, San Diego, CA, USA).