Fig. 1

MDA5 suppresses LINE-1 activity in an EGFP-based LINE-1 retrotransposition assay. A LINE-1 expressing cassettes used in this study. In the retrotransposition-competent plasmid L1-RPS, an anti-sense CMV-EGFP cassette is inserted into the 3′-UTR of LINE-1, whereas the EGFP gene is interrupted by a sense Group I intron. JM111 is based on L1-RPS but contains an R260A/R261A mutation on ORF1p, and is therefore incompetent in retrotransposition. B Schematic representation of the mechanism of the EGFP-based LINE-1 assay. To produce an EGFP signal after transfection, L1-RPS needs to be transcribed through the LINE-1 5′-UTR promoter to generate the RNA, spliced to remove the intron, and then reverse-transcribed and integrated into the genome to finish the replication. Only then can the EGFP signal be generated through the expression of the integrated CMV-EGFP cassette. Any direct transcription of the CMV-EGFP cassette from the plasmid will not produce EGFP because of the un-spliced intron. C Flow cytometry results showing that MDA5-myc potently suppresses the retrotransposition activity of L1-RPS in HEK293T cells. HEK293T cells seeded on a 24-well plate were co-transfected with 1 μg of L1-RPS and control vector VR1012 (225 ng) or MDA5-myc-expressing vector (25 ng, 75 ng, and 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. D Flow cytometry results showing that MDA5-myc does not suppress EGFP expression driven by the CMV promoter. HEK293T cells seeded on a 24-well plate were co-transfected with 45 ng of pEGFP-C1 and 225 ng of control vector VR1012 or 25, 75, or 225 ng of MDA5-myc-expressing vector. Cells were collected at 96 h post-transfection to detect EGFP-positive cells through flow cytometry. Western blotting results above indicate the MDA5-myc protein levels in transfected cells. E qRT-PCR results showing efficacy of IFIH1-specific siRNAs in HEK293T cells. HEK293T cells seeded on a 24-well plate were transfected with control 100 nM (final concentration) non-targeting control siRNA (siNC) or IFIH1-specific siRNAs (siIFIH1–1 and siIFIH1–2). Total RNA was extracted for each sample at 72 h post-transfection, and levels of endogenous IFIH1 mRNA (encoding MDA5) were determined. F Flow cytometry results showing that LINE-1 activity is potently increased in HEK293T cells treated with IFIH1-specific siRNAs. HEK293T cells seeded on a 24-well plate were first treated with 100 nM siNC, siIFIH1–1 or siIFIH1–2, and then transfected with 1 μg of L1-RPS after 24 h. Cells were collected at 96 h post-transfection to detect EGFP-positive cells through flow cytometry. G Flow cytometry results showing that MDA5-myc potently suppresses the retrotransposition activity of L1-RPS in HeLa cells. HeLa cells seeded on a 24-well plate were co-transfected with 1 μg of L1-RPS and control vector VR1012 (225 ng) or MDA5-myc-expressing vector (25 ng, 75 ng, and 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