Generation of SupT1 cell lines stably expressing CD46 (S201L‐BC1)

Lentiviral particles were produced as previously described [20]. In brief, HEK293T cells were co‐transfected with 1 μg pRSV‐REV, 2 μg pMD.2G, 2 μg pMDLg/p‐RRE, and 3 μg pCCL‐WPS‐PGK‐CD46‐puro‐WHV vector, using FuGENE HD Transfection Reagent (Promega Corporation, USA). Approximately 24 h prior to transfection, 8 × 10⁶ HEK293T cells were plated to 40–50% confluence. Transfection was done according to the manufacturer's protocol, and culture medium was collected at 48 and 72 h post transfection. The collected medium was filtered through a 450 nM filter and concentrated using 20% sucrose solution by a 2 h ultracentrifugation at 25.000 × g, 4°C. The lentiviral particles were resuspended in PBS using three cycles of vortexing (15 s) and incubated on ice (2 min). The stocks were kept at −80°C. SupT1^ΔCD46 cells (generated as described elsewhere [20]) were adjusted to 0.5 × 10⁶ cells/mL 24 h prior to transduction. For the generation of stable cell lines, 600.000 SupT1^ΔCD46 cells (0.522 × 10⁶ cells/mL) were transduced with different amounts of lentivirus in the presence of 25 μg/mL protamine sulfate (Sigma‐Aldrich), using a 6‐well plate. 45 h post transduction, the cells were selected with 2 μg/mL puromycin (Gibco) for a minimum of 14 days. Cell lines were isolated upon transduction with two separate lentivirus preparations, obtained with separate DNA constructs, i.e., S201L.1 and S201L.2. The established cell lines were analyzed using flow cytometry, allowing an evaluation of the CD46 expression.