Evaluating Ago2 Knockout Efficiency

To assess the Ago2 knockout efficiency, AF525 and AF5 cells were seeded in a 24-well plate (1.5 × 10⁵ cells/well). After 24 h, cells were transfected using 2 µL of DharmaFECT 2 (Horizon Discovery Ltd., Cambridge, UK) per well. Sixty nanograms of pIZ-Fluc and 10 ng pAcIE1-Rluc were co-transfected either with 1 ng siRNA (targeting FFluc or non-specific hygromycin B resistance gene as control [19 (link)]) or 10 ng dsRNA (targeting Rluc or eGFP (control)). For Ago2 reconstitution assays, AF5 and AF525 cells at a density of 2.4 × 10⁵ cells per well in poly-l-lysine-coated wells were transfected 24 h after seeding using 2 µL of DharmaFECT 2 (Horizon Discovery Ltd., Cambridge, UK) per well. Cells received 100 ng of pIZ-Fluc and 100 ng pAcIE1-Rluc, which were co-transfected with 10 ng dsRNA (targeting FFluc or non-specific lacZ). Additionally, cells were co-transfected with 500 ng per well plasmids expressing either myc-Ago2 or myc-eGFP (control). At 48 h post transfection (hpt), cells were lysed, and relative luciferase activity was measured using Dual-Luciferase Reporter Assay System (Promega Corp., Fitchburg, WI, USA).