Fig. 3

IFNβ elevation contributes mildly to MDA5-mediated LINE-1 suppression. A Flow cytometry data suggesting that exogenous MDA5-myc suppresses L1-RPS retrotransposition without significantly enhance endogenous IFNβ expression. HEK293T cells seeded on a 24-well plate were co-transfected with 1 μg of L1-RPS and 225 ng of control vector VR1012 or 25, 75, or 225 ng of MDA5-myc- or IFNβ-expressing vector. Cells were collected at 96 h post-transfection to detect EGFP-positive cells through flow cytometry. Western blotting results above indicate IFNβ and MDA5 protein levels in transfected cells. B Schematic representation of the IFNB-Luc expressing cassette (in the backbone vector pGL3-Basic), where the expression of firefly luciferase is driven by the IFNB promoter. C and E Luciferase activity data indicating the potency of wild-type MDA5-myc or its mutants in IFNβ elevation. HEK293T cells seeded in a 24-well plate were co-transfected with 100 ng of IFNB-Luc and 45 ng of control vector VR1012 or one of MDA5-myc-expressing vectors (5, 15, or 45 ng). Luciferase activity was tested at 48 h post-transfection. The western blotting results above indicate the MDA5 protein levels in transfected cells. D and F Flow cytometry results showing the efficacy of MDA5 mutants in LINE-1 suppression. HEK293T cells seeded on a 24-well plate were co-transfected with 1 μg of L1-RPS and control vector VR1012 (225 ng) or one of MDA5-myc-expressing vectors (25, 75, 225 ng). Cells were collected at 96 h post-transfection to detect EGFP-positive cells through flow cytometry. The western blotting results above indicate the MDA5-myc protein levels in transfected cells