Fig. S4.

RNA immunoprecipitation assay showed that the tMDA5- or tMDA5/tLGP2-associated RNAs are SeV RNAs. (A) Sequencing electrophoregrams showing the MDA5- or tMDA5/tLGP2-associated RNA as the SeV RNA. The tMDA5 or tMDA5/tLGP2 immunoprecipitates were extracted for RNA and were reversely transcribed to cDNA, followed by PCR amplification and sequencing. The sequencing results matched to the SeV genome fragment (GenBank accession no. M30202.1). Input Sev RNA: viral RNA isolated from whole cell lysate infected with SeV. IP SeV RNA: viral RNA isolated from the immunoprecipitated protein-RNA complexes. (B) Quantification of the amount of EMCV RNA in hRIG-I, tMDA5, and IgG immunoprecipitates by using the strand-specific RT-PCR. (Top) Immunoblot showing Flag-tagged hRIG-I and tMDA5 in IP. (Bottom) The EMCV RNA levels were calculated by comparison with a dilution curve of cDNA prepared from the EMCV-infected cells. (C) Up-regulation of the tIFNB1 mRNA expression by the tMDA5-associated EMCV RNAs was dependent on tMDA5. TSPRCs (1 × 10⁵) were first transfected with sitMDA5 (50 nM) and siRNA control (Scramble, 50 nM) for 24 h and then transfected with the indicated amount EMCV RNAs for 9 h before the harvest. The relative tIFNB1 mRNA levels were measured by using qRT-PCR. (D) Effects of the overexpression of the tLGP2 and tMDA5 on the induction of the IFN-β-Luc reporter in TSPRCs. Cells (1 × 10⁴ cells per well) were grown in 24-well plate overnight and were transfected with the IFN-β-Luc reporter vector (100 ng) and TK (10 ng, as an inner control). The tMDA5 expression vector was transfected at a constant quantity of 50 ng plasmid per well, whereas the amount of tLGP2 was titrated. After 24 h, cells were transfected with low-molecular-weight poly I:C (1 μg/mL) or without low-molecular-weight poly I:C (Control) for 12 h before the harvest for luciferase assay. (E) Both high-molecular-weight poly I:C (poly I:C H) and low molecular weight poly I:C (poly I:C L) up-regulated the tIFNB1 mRNA levels in TSPRCs. The TSPRCs (1 × 10⁵) were transfected with poly I:C H (100 ng/mL) or poly I:C L (100 ng/mL) or without transfection (NC) and were harvested at the indicated time points after transfection. (F) Knockdown of tLGP2 (sitLGP2) or tMDA5 (sitMDA5) caused an inhibition of poly I:C-induced tIFNB1 mRNA expression. The TSPRCs (1 × 10⁵/well) were grown in 12-well plate overnight and were transfected with the siRNA negative control (Scramble, 50 nM) or indicated siRNA (50 nM) for 36 h, followed by transfection with poly I:C H or poly I:C L (100 ng/mL) for 12 h, respectively, before the harvest. The relative tIFNB1 mRNA levels were measured using qRT-PCR. *P < 0.05, **P < 0.01, ***P < 0.001, relative to the NC or Scramble, Student t test. Bars represent mean ± SEM. All experiments were repeated for three times with similar results.