Fig. 6

The 2CARD region is critical for MDA5-mediated LINE-1 suppression. A Schematic representation showing the deletion mutants of MDA5. B and C Luciferase activity data indicating the potency of MDA5-myc mutants in LINE-1 5′-UTR regulation. HEK293T cells seeded in a 24-well plate were co-transfected with 200 ng of 5’UTR-Luc and control vector VR1012 (225 ng) or one of MDA5-myc-expressing vectors (25, 75, or 225 ng). Luciferase activity was tested at 48 h post-transfection. The western blotting results show the MDA5-myc protein levels in the transfected cells. D and E Flow cytometry results showing the efficacy of MDA5-myc 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, or 225 ng), and were collected at 96 h post-transfection to detect EGFP-positive cells through flow cytometry. The western blotting results indicate the MDA5-myc protein levels in the transfected cells. F Co-IP experiment results indicating that MDA5 can interact with 5UTR-Luc plasmid DNA. HEK293T cells seeded on 6-well plate were co-transfected with 5UTR-Luc (800 ng) and another vector expressing YY1-myc (1200 ng), MDA5-myc (1200 ng), Trunc1 (1200 ng), Trunc4 (1200 ng), or the control vector VR1012 (1200 ng). Co-IP was performed at 48 h post transfection. DNA was extracted from eluted samples and subjected to qRT-PCR with primers targeting the luciferase gene. The multi-band phenomena detected for MDA5 in panels B-F are similar to previous reported observations [37]. Arrows are used to indicate bands of interested proteins with predicted sizes for YY1-myc, wild type MDA5-myc, and its mutants in these panels