Journal: Scientific Reports

Article Title: Chemokine profiles of interstitial pneumonia in patients with dermatomyositis: a case control study

doi: 10.1038/s41598-017-01685-5

Figure Lengend Snippet: Comparison of each of the chemokine levels between anti-MDA5 antibody-positive and -negative cases. CCL: C-C motif chemokine ligand; CXCL: C-X-C motif chemokine ligand; negative: anti-MDA5 antibody-negative patients; positive: anti-MDA5 antibody-positive patients. The P value was estimated by the Mann-Whitney U-test. * P < 0.05.

Article Snippet: Anti-MDA5 antibody was determined by ELISA using recombinant MDA5 antigen (OriGene, Rockville, MD, USA) as described previously .

Techniques: Comparison, MANN-WHITNEY

Journal: bioRxiv

Article Title: Inhibiting DDX3X triggers tumor-intrinsic type I interferon response and enhances anti-tumor immunity

doi: 10.1101/2020.09.09.289587

Figure Lengend Snippet: a , Western blot analysis of dsDNA sensor and downstream target, cGAS and STING, in DDX3X-control or -depleted MCF7 or MDA-MB-453 cells. b , Western blot analysis of dsRNA sensors and downstream target, TLR3, PKR and elF2a, in DDX3X-control or -depleted MCF7 or -MDA-MB-453 cells. c , Western blot analysis of dsRNA sensors, RIG-I, MDA5, and OAS1, in DDX3X-control or - depleted MCF7 or MDA-MB-453 cells. d , Western blot analysis of phosphorylation of transcription factors, NFκB, IRF3, and IRF7, in DDX3X-control or -depleted MCF7 or MDA-MB-453 cells. e , qRT-PCR of IFNB1 in DDX3X-control or -KD MCF7 following by MDA5 knockout. f , Western blot analysis of phosphorylated and total NFκB, STAT1, and STAT2 in DDX3X wildtype and DDX3X f/f MEF cells after Ad-Cre virus treatment. g , Western blot analysis of STAT1 and STAT2 in shRNA-mediated DDX3X KD MEF cells. h , qRT-PCR of IFNB1 in DDX3X f/f MEF cells 4 days after Ad-Cre virus treatment. i , Schema of the dsRNA-mediated type I IFN signaling in DDX3X-depleted cancer and MEF cells. Data are representative of three independent experiments. Data are shown as mean ± SEM. Statistics were calculated using unpaired t-tests. * P < 0.05; ** P < 0.01; ns, not significant.

Article Snippet: The IFIH1 gene was deleted using a MDA5 (IFIH1) Human Gene Knockout Kit (OriGene, KN415661) according to the manufacturer’s instructions with target sequences (5’-CTGGATGTACATTTTCACCC-3’).

Techniques: Western Blot, Control, Phospho-proteomics, Quantitative RT-PCR, Knock-Out, Virus, shRNA

Journal: bioRxiv

Article Title: ISG15-dependent Activation of the RNA Sensor MDA5 and its Antagonism by the SARS-CoV-2 papain-like protease

doi: 10.1101/2020.10.26.356048

Figure Lengend Snippet: (a, b) ELISA of IFN-β from supernatants of MEFs (WT or Isg15 −/− ) (a) and HeLa cells (WT or ISG15 KO) (b) transiently transfected with increasing amounts of FLAG-tagged MDA5 or RIG-I for 40 h. Whole cell lysates (WCLs) were probed by immunoblotting (IB) with anti-ISG15, anti-FLAG, and anti-Actin (loading control). (c) ELISA of IFN-β from supernatants of WT or Isg15 −/− MEFs that were mock-stimulated or transfected with EMCV-RNA (0.1 or 0.4 μg/mL), HMW-poly (I:C) (0.5 μg/mL), or RABV Le (1 pmol/mL), or infected with SeV (10 HAU/mL) for 24 h. (d) Quantitative RT-PCR (qRT-PCR) analysis of IFNB1 and CCL5 mRNA in WT and Isg15 −/− MEFs stimulated as in (c). (e) IRF3 phosphorylation in the WCLs of NHLFs that were transfected with the indicated siRNAs for 30 h and then mock-stimulated or transfected with EMCV-RNA (0.4 μg/mL) or RABV Le (1 pmol/mL) for 6 h, assessed by IB with anti-pS396-IRF3 and anti-IRF3. (f) ELISA of IFN-β from supernatants of NHLFs that were transfected with the indicated siRNAs for 30 h and then mock-stimulated or transfected with EMCV-RNA (0.4 μg/mL) or RABV Le (1 pmol/mL), or infected with SeV (10 HAU/mL) for 16 h. (g) ELISA of IFN-β from the supernatants of PBMCs that were transduced for 40 h with the indicated shRNAs and then infected with mutEMCV (MOI 10) or SeV (200 HAU/mL) for 8 h. (h) qRT-PCR analysis of IFNA2 and IL-6 mRNA in PBMCs that were transduced and infected as in (g). Data are representative of at least two independent experiments (mean ± s.d. of n = 3 biological replicates in a, b, c, d, f and mean of n = 2 biological replicates in g and h). * p < 0.05, ** p < 0.01, *** p < 0.001 (unpaired Student’s t -test). ND, not detected; NS, not significant.

Article Snippet: Scrambled shRNA control lentiviral particles and shRNA lentiviral particles targeting ISG15 (TL319471V) or IFIH1 (TL303992V) were purchased from OriGene.

Techniques: Enzyme-linked Immunosorbent Assay, Transfection, Western Blot, Control, Infection, Quantitative RT-PCR, Phospho-proteomics

Journal: bioRxiv

Article Title: ISG15-dependent Activation of the RNA Sensor MDA5 and its Antagonism by the SARS-CoV-2 papain-like protease

doi: 10.1101/2020.10.26.356048

Figure Lengend Snippet: (a) Endogenous MDA5 ISGylation in NHLFs that were mock-treated, transfected with HMW-poly (I:C) (0.1 μg/mL) for 40 h (left), or infected with DENV or ZIKV (MOI 1 for each) for 48 h (right), determined by immunoprecipitation (IP) with anti-MDA5 (or an IgG isotype control) followed by IB with anti-ISG15 and anti-MDA5. WCLs were probed by IB with anti-ISG15 and anti-Actin (loading control). (b) ISGylation of FLAG-tagged MDA5–2CARD and MDA5ΔCARD in transiently transfected HEK293T cells that also expressed V5-ISG15, HA-Ube1L, and FLAG-UbcH8, assessed by FLAG pulldown (PD) and IB with anti-V5 and anti-FLAG forty hours after transfection. WCLs were probed by IB with anti-HA, anti-FLAG, anti-V5, and anti-Actin. (c) Endogenous MDA5 ISGylation in ISG15 KO HeLa cells stably reconstituted with vector, WT ISG15 or ISG15-AA and co-transfected with HA-Ube1L and FLAG-UbcH8 after IFN-β treatment (1,000 U/mL) for 24 h, determined by IP with anti-MDA5 and IB with anti-ISG15 and anti-MDA5. (d) ISGylation of GST-MDA5–2CARD WT and K23R/K43R in HEK293T cells that were co-transfected with V5-ISG15, HA-Ube1L, and FLAG-UbcH8 for 24 h, determined by GST-PD and IB with anti-V5 and anti-GST. (e) ISGylation of FLAG-tagged MDA5 WT and K23R/K43R in HEK293T cells that were co-transfected with V5-ISG15, HA-Ube1L, and FLAG-UbcH8, determined by FLAG-PD and IB with anti-V5 and anti-FLAG. (f) IFN-β-luciferase reporter activity in HEK293T cells that were transfected for 40 h with vector, or FLAG-tagged MDA5 WT or mutants. Luciferase activity is presented as fold induction relative to the values for vector-transfected cells, set to 1. WCLs were probed by IB with anti-FLAG and anti-Actin. (g) qRT-PCR analysis of IFNB1 and CCL5 mRNA in HEK293T cells that were transiently transfected with either vector, or increasing amounts of FLAG-tagged MDA5 WT or K23R/K43R. (h) STAT1 phosphorylation and ISG (IFIT1 and 2) protein abundance in the WCLs of HEK293T cells that were transiently transfected with vector or FLAG-tagged MDA5 WT or K23R/K43R, determined by IB with anti-pY701-STAT1, anti-STAT1, anti-IFIT1, anti-IFIT2, anti-FLAG (expression control) and anti-Actin (loading control). (i) qRT-PCR analysis of IFNB1 , CCL5 , OAS1 , and RSAD2 mRNA in MDA5 KO SVGAs that were reconstituted with either empty vector or FLAG-tagged MDA5 WT, K23R/K43R or S88E. Data are representative of at least two independent experiments (mean ± s.d. of n = 3 biological replicates in f, g, and i). * p < 0.05, ** p < 0.01, *** p < 0.001 (unpaired Student’s t -test). NS, not significant.

Article Snippet: Scrambled shRNA control lentiviral particles and shRNA lentiviral particles targeting ISG15 (TL319471V) or IFIH1 (TL303992V) were purchased from OriGene.

Techniques: Transfection, Infection, Immunoprecipitation, Control, Stable Transfection, Plasmid Preparation, Luciferase, Activity Assay, Quantitative RT-PCR, Phospho-proteomics, Quantitative Proteomics, Expressing

Journal: bioRxiv

Article Title: ISG15-dependent Activation of the RNA Sensor MDA5 and its Antagonism by the SARS-CoV-2 papain-like protease

doi: 10.1101/2020.10.26.356048

Figure Lengend Snippet: (a , b) Cytosol-mitochondria fractionation of WCLs from NHLFs that were transfected for 30 h with non-targeting control siRNA (si.C) or ISG15-specific siRNA (si.ISG15) and then mock-treated or transfected with EMCV-RNA (0.4 μg/mL) (a) or RABV Le (1 pmol/mL) (b) for 16 h. IB was performed with anti-MDA5 (a), anti-RIG-I (b), anti-ISG15 and anti-Actin (a, b). α-Tubulin and MAVS served as purity markers for the cytosolic and mitochondrial fraction, respectively (a, b). (c) Endogenous MDA5 oligomerization in WT and Isg15 −/− MEFs that were transfected with EMCV-RNA (0.5 μg/mL) for 16 h, assessed by SDD-AGE and IB with anti-MDA5. WCLs were further analyzed by SDS-PAGE and probed by IB with anti-MDA5 and anti-Actin. (d) Oligomerization of FLAG-MDA5–2CARD in HEK293T cells that were transfected with the indicated siRNAs together with or without HA-Ube1L and FLAG-UbcH8 for 48 h, determined by native PAGE and IB with anti-FLAG. WCLs were further analyzed by SDS-PAGE and probed by IB with anti-FLAG, anti-HA, anti-ISG15, and anti-Actin. (e) Oligomerization of FLAG-MDA5 WT and K23R/K43R in transiently transfected MDA5 KO HEK293 cells, assessed by SDD-AGE and IB with anti-FLAG. WCLs were further analyzed by SDS-PAGE and IB with anti-FLAG and anti-Actin. (f) Oligomerization of FLAG-tagged MDA5 WT and mutants in transiently transfected MDA5 KO HEK293 cells, assessed by native PAGE and IB with anti-MDA5. WCLs were further analyzed by SDS-PAGE and probed by IB with anti-MDA5 and anti-Actin. (g) IFN-β-luciferase reporter activity in MDA5 KO HEK293 cells that were transfected for 24 h with either empty vector, or FLAG-tagged MDA5 WT or mutants. Luciferase activity is presented as fold induction relative to the values for vector-transfected cells, set to 1. Data are representative of at least two independent experiments (mean ± s.d. of n = 3 biological replicates in f). *** p < 0.001 (unpaired Student’s t -test).

Article Snippet: Scrambled shRNA control lentiviral particles and shRNA lentiviral particles targeting ISG15 (TL319471V) or IFIH1 (TL303992V) were purchased from OriGene.

Techniques: Fractionation, Transfection, Control, SDS Page, Clear Native PAGE, Luciferase, Activity Assay, Plasmid Preparation

Journal: bioRxiv

Article Title: ISG15-dependent Activation of the RNA Sensor MDA5 and its Antagonism by the SARS-CoV-2 papain-like protease

doi: 10.1101/2020.10.26.356048

Figure Lengend Snippet: (a) EMCV titers in the supernatant of HEK293T cells that were transiently transfected for 40 h with either empty vector, or FLAG-tagged MDA5 WT or K23/K43R and then infected with EMCV (MOI 0.001) for 24 h, determined by TCID50 assay. (b) Percentage of DENV-infected MDA5 KO HEK293 cells that were transiently transfected for 24 h with either empty vector or FLAG-tagged MDA5 WT or K23R/K43R and then mock-treated or infected with DENV (MOI 5) for 48 h, assessed by FACS using an anti-flavivirus E (4G2) antibody. SSC, side scatter. (c) ZIKV titers in the supernatant of MDA5 KO SVGAs that were transiently transfected for 30 h with either empty vector, or FLAG-tagged MDA5 WT, K23R/K43R, or S88E and then infected with ZIKV (MOI 0.1) for the indicated times, determined by plaque assay. (d) SCoV2 titers in the supernatant of HEK293T-hACE2 cells that were transiently transfected for 24 h with either empty vector, or FLAG-tagged MDA5 WT or K23/K43R and then infected with SCoV2 (MOI 0.5) for 24 h, determined by plaque assay. (e) Schematic of the experimental approach to decouple the role of ISG15 in MDA5-mediated IFN induction from its role in dampening IFNAR signaling. (f) NHLF ‘donor’ cells were transfected for 40 h with the indicated siRNAs and then infected with mutEMCV (MOI 0.1) for 16 h. Cell culture supernatants were UV-inactivated and transferred onto Vero ‘recipient’ cells for 24 h, followed by infection of cells with ZIKV (MOI 0.002 to 2) for 72 h. ZIKV-positive cells were determined by immunostaining with anti-flavivirus E (4G2) antibody and visualized using the KPL TrueBlue peroxidase substrate. (g) RIG-I KO HEK293 ‘donor’ cells were transfected for 24 h with si.C or si.ISG15 and subsequently transfected with either empty vector or FLAG-tagged MDA5 WT or K23R/K43R for 24 h, followed by EMCV infection (MOI 0.001) for 16 h. UV-inactivated culture supernatants were transferred onto Vero ‘recipient’ cells for 24 h, followed by infection with EMCV (MOI 0.001 to 0.1) for 40 h. EMCV-induced cytopathic effects were visualized by Coomassie blue staining. Data are representative of at least two independent experiments (mean ± s.d. of n = 3 biological replicates in a, b, c). ** p < 0.01 (unpaired Student’s t -test).

Article Snippet: Scrambled shRNA control lentiviral particles and shRNA lentiviral particles targeting ISG15 (TL319471V) or IFIH1 (TL303992V) were purchased from OriGene.

Techniques: Transfection, Plasmid Preparation, Infection, TCID50 Assay, Plaque Assay, Cell Culture, Immunostaining, Staining

Journal: bioRxiv

Article Title: ISG15-dependent Activation of the RNA Sensor MDA5 and its Antagonism by the SARS-CoV-2 papain-like protease

doi: 10.1101/2020.10.26.356048

Figure Lengend Snippet: (a) Ribbon representation of the crystal structure of the SCoV2 PLpro: ISG15 complex (PDB: 6YVA). Key residues that mediate ‘site 1’ interaction (N156 and R166/E167) or ‘site 2’ interaction (F69) in PLpro, as well as its catalytically-active site (C111), are indicated. (b) ISGylation of GST-MDA5–2CARD in HEK293T cells that were co-transfected for 20 h with vector or V5-tagged SCoV2 PLpro WT or mutants, along with FLAG-ISG15, HA-Ube1L, and FLAG-UbcH8, determined by GST-PD and IB with anti-FLAG and anti-GST. WCLs were probed by IB with anti-V5, anti-HA, anti-FLAG, and anti-Actin. (c) Binding of HA-tagged MDA5 or RIG-I to V5-SCoV2-PLpro or FLAG-MeV-V (control) in transiently transfected HEK293T cells, determined by HA-PD and IB with anti-V5 or anti-FLAG, and anti-HA. WCLs were probed by IB with anti-V5 and anti-FLAG. (d) Oligomerization of FLAG-MDA5–2CARD in HEK293T cells that were co-transfected with vector, or V5-SCoV2 PLpro WT or C111A for 24 h, assessed by Native PAGE and IB with anti-FLAG. WCLs were further analyzed by SDS-PAGE and probed by IB with anti-FLAG, anti-V5 and anti-Actin. (e) ISGylation of GST-MDA5–2CARD in HEK293T cells that also expressed FLAG-ISG15, HA-Ube1L and FLAG-UbcH8, and were co-transfected for 40 h with vector or the indicated V5-tagged coronaviral PLpro, determined by GST-PD and IB with anti-FLAG, anti-V5, and anti-GST. Data are representative of at least two independent experiments.

Article Snippet: Scrambled shRNA control lentiviral particles and shRNA lentiviral particles targeting ISG15 (TL319471V) or IFIH1 (TL303992V) were purchased from OriGene.

Techniques: Transfection, Plasmid Preparation, Binding Assay, Control, Clear Native PAGE, SDS Page

Journal: bioRxiv

Article Title: ISG15-dependent Activation of the RNA Sensor MDA5 and its Antagonism by the SARS-CoV-2 papain-like protease

doi: 10.1101/2020.10.26.356048

Figure Lengend Snippet: (a) qRT-PCR analysis of IFNB1 , IFNL1 , ISG15 , MDA5 , and RIG-I transcripts in NHLFs that were transfected with the indicated siRNAs for 40 h and then transfected with mock-RNA or SCoV2-RNA (0.4 μg/mL) for 24 h. (b) Binding of SCoV2 Nsp3 to endogenous MDA5 in A549-hACE2 cells that were infected with SCoV2 (MOI 0.5) for 24 h, determined by IP with anti-MDA5 (or an IgG isotype control) followed by IB with anti-PLpro and anti-MDA5. WCLs were probed by IB with anti-PLpro (Nsp3) and anti-Actin. (c) Endogenous MDA5 ISGylation in A549-hACE2 cells that were mock-infected, or infected with SCoV2 or DENV (MOI 0.1 for each) for 48 h, determined by immunoprecipitation (IP) with anti-MDA5 followed by IB with anti-ISG15 and anti-MDA5. Protein abundance of IFIT1, RSAD2, IFITM3, ISG15 and actin in the WCLs were probed by IB. Efficient virus replication was verified by immunoblotting WCLs with anti-PLpro (Nsp3) or anti-NS3 (DENV). (d) qRT-PCR analysis of IFNB1 , CCL5 , IFIT1 transcript, and EMCV genomic RNA (gRNA) in HeLa cells that were transiently transfected for 24 h with vector, or V5-SCoV2 PLpro WT or mutants and then infected with mutEMCV (MOI 0.5) for 12 h. (e) EMCV titers in the supernatant of RIG-I KO HEK293 cells that were transiently transfected for 24 h with vector or FLAG-MDA5 along with V5-SCoV2 PLpro WT, C111A, or R166S/E167R and then infected with EMCV (MOI 0.001) for 16 h, determined by plaque assay. (f) Protein abundance of the indicated ISGs in the WCLs from the experiment in (e), determined by IB with the indicated antibodies. Data are representative of at least two independent experiments (mean ± s.d. of n = 3 biological replicates in a, d, e). * p < 0.05, *** p < 0.001 (unpaired Student’s t -test).

Article Snippet: Scrambled shRNA control lentiviral particles and shRNA lentiviral particles targeting ISG15 (TL319471V) or IFIH1 (TL303992V) were purchased from OriGene.

Techniques: Quantitative RT-PCR, Transfection, Binding Assay, Infection, Control, Immunoprecipitation, Quantitative Proteomics, Virus, Western Blot, Plasmid Preparation, Plaque Assay

Journal: eLife

Article Title: An arms race between 5’ppp-RNA virus and its alternative recognition receptor MDA5 in RIG-I-lost teleost fish

doi: 10.7554/elife.94898

Figure Lengend Snippet: Figure 1. A genetic compensation response model for RIG loss. (A) Loss of RIG-I in vertebrates. Each branch tip represents one species. Red lines show lineages where loss of RIG-I. The red circle indicates the occurrence of an independent loss event. Branch lengths scale in millions of years (MYA). (B) Comparative analysis of gene synteny of RIG-I in vertebrate genomes. (C and D) SCRV-RNA and VSV-RNA were extracted from SCRV and VSV virus and dephosphorylated them with Calf Intestinal Alkaline Phosphatase (CIAP). Then, MKC cells were transfected with SCRV-RNA and SCRV-RNA +CIAP, and DF-1 cells were transfected with VSV-RNA and VSV-RNA +CIAP. IRF3 dimerization was analyzed by native gel electrophoresis. (E) MKC cells were transfected with different concentrations of SCRV-RNA and SCRV-RNA +CIAP, then the expression of IFN-1 was detected by qRT-PCR. As a control, the expression level of IFN-1 in MKC cells transfected with the same volume of water was set to ‘1’. (F) Predicted protein structures of MDA5 and LGP2 of M. miiuy and RIG-I of H. sapiens. (G) The expression of MDA5 and LGP2 plasmids of M. miiuy were detected by western Blot. (H) MKC cells were co- transfected with MDA5 plasmids and LGP2 plasmids for 24 hr and then treated with SCRV (MOI = 5) for 24 hr. The expression of IFN-1 was determined by qPCR. (I) MKC cells were co-transfected with MDA5 plasmids and si-LGP2 for 24 hr and then treated with SCRV (MOI = 5) for 24 hr. The expression of LGP2 and IFN-1 was determined by qPCR. (J) A genetic compensation response model for RIG loss. All data presented as the means ± SE from three independent triplicated experiments. **, p<0.01, as determined by Student’s t test.

Article Snippet: The antibody against MDA5 was diluted at 1: 500 (Beyotime, Cat# AF7164); The antibody against IRF3 was diluted at 1: 500 (Boster, Cat# BA4351- 2); anti- Flag, anti- Myc, anti- HA and anti- Tubulin monoclonal antibody were diluted at 1: 1,000 (Beyotime); and HRP- conjugated anti- rabbit IgG or anti- mouse IgG (Abbkine) at 1: 5,000.

Techniques: Virus, Transfection, Nucleic Acid Electrophoresis, Expressing, Quantitative RT-PCR, Control, Western Blot

Journal: eLife

Article Title: An arms race between 5’ppp-RNA virus and its alternative recognition receptor MDA5 in RIG-I-lost teleost fish

doi: 10.7554/elife.94898

Figure Lengend Snippet: Figure 2. MDA5 promotes host antiviral innate immunity. (A) Silence efficiency of si-MDA5 measured by qRT-PCR and Western blotting. Two siRNAs of MDA5 were transfected into MPC cells for 48 hr, respectively. (B) MKC cells were transfected with MDA5 plasmids for 48 hr, then the overexpression efficiency was detected through MDA5 endogenous antibodies. (C) Subcellular localization of MDA5 in MKCs by immunofluorescence. (D) MDA5 knockdown suppresses NF-κB, IRF3, IRF7, and IFN-1 signaling. MPC cells were transfected with pRL-TK Renilla luciferase plasmid, luciferase reporter genes, together with MDA5 expression plasmid. At 48 hr post-transaction, the luciferase activity was measured and normalized to renilla luciferase activity. (E) Knockdown of MDA5 attenuates the expression of antiviral genes. MPC cells were transfected with si-Ctrl or si-MDA5 for 24 hr and then treated with SCRV (MOI = 5) or poly(I:C)-HMW for 24 hr. The expression of IFN-1, Mx1, ISG15, and Viperin were determined by qPCR. (F and G) Overexpression of MDA5 promotes the expression of antiviral genes. MKC cells were transfected with vector or MDA5 expression plasmids for 24 hr and then treated with SCRV (MOI = 5) or poly (I:C)-HMW for 24 hr. The expression of MDA5 (F), and antiviral genes, including IFN-1, Mx1, ISG15, and Viperin (G) were determined by qPCR. (H and J) MPC cells transfected with si-Ctrl or si-MDA5 and MKC cells transfected with pcDNA3.1 vector or MDA5 plasmid 24 hr and then treated with SCRV at the dose indicated for 48 hr. Then, cell monolayers were fixed with 4% paraformaldehyde and stained with 1% crystal violet. (I and K) MDA5 inhibits SCRV replication. MPC cells were transfected with si-Ctrl or si-MDA5 and MKC cells were transfected with pcDNA3.1 vector or MDA5 expression plasmid for 24 hr, then infected with SCRV (MOI = 5) for 12, 24, or 48 hr. The qPCR analysis was conducted for SCRV-M and SCRV-G RNA levels. (L and M) MDA5 immunoprecipitates with MAVS (L) and STING (M). EPC cells (~1 × 107) were co- transfected with MAVS-Myc or STING-Myc and MDA5-Flag expression plasmids for 24 hr, followed by immunoprecipitation (IP) with anti-Myc. (N and O) MKC cells were transfected with MDA5 plasmids and STING plasmids (N) or si-STING (O) for 24 hr and then treated with SCRV (MOI = 5) for 24 hr. The expression of IFN-1 was determined by qPCR. (P and Q) MKC cells were transfected with si-MDA5 for 24 hr and then transfected with SCRV-RNA

Article Snippet: The antibody against MDA5 was diluted at 1: 500 (Beyotime, Cat# AF7164); The antibody against IRF3 was diluted at 1: 500 (Boster, Cat# BA4351- 2); anti- Flag, anti- Myc, anti- HA and anti- Tubulin monoclonal antibody were diluted at 1: 1,000 (Beyotime); and HRP- conjugated anti- rabbit IgG or anti- mouse IgG (Abbkine) at 1: 5,000.

Techniques: Quantitative RT-PCR, Western Blot, Transfection, Over Expression, Immunofluorescence, Knockdown, Luciferase, Plasmid Preparation, Expressing, Activity Assay, Staining, Infection, Immunoprecipitation

Journal: eLife

Article Title: An arms race between 5’ppp-RNA virus and its alternative recognition receptor MDA5 in RIG-I-lost teleost fish

doi: 10.7554/elife.94898

Figure Lengend Snippet: Figure 3. RD domain is required for MDA5 to recognize SCRV RNA. (A) The association of MDA5 proteins with SCRV. (B) Schematics and expression of MDA5 and truncated MDA5 (MDA5-△RD). (C) The association of MDA5 and MDA5-△RD proteins with SCRV. (D) MKC cells transfected with pcDNA3.1 vector, MDA5, or MDA5-△RD plasmids for 24 hr and then treated with SCRV at the dose indicated for 48 hr. Then, cell monolayers were fixed with 4% paraformaldehyde and stained with 1% crystal violet. (E) MKC cells were transfected with pcDNA3.1 vector, MDA5 or MDA5-△RD expression plasmids for 24 hr, then infected with SCRV for 24 hr, then the qPCR analysis was conducted for SCRV-M and SCRV-G RNA levels. (F–I) Expression of IFN-1 (F), Mx1 (G), ISG15 (H), and Viperin (I) in Mock-infected, SCRV-infected, and poly(I:C)-HMW-stimulated MKC cells transfected with pcDNA3.1 vector, MDA5, or MDA5-△RD expression plasmids. All data presented as the means ± SE from three independent triplicated experiments. **, p<0.01, as determined by Student’s t test.

Article Snippet: The antibody against MDA5 was diluted at 1: 500 (Beyotime, Cat# AF7164); The antibody against IRF3 was diluted at 1: 500 (Boster, Cat# BA4351- 2); anti- Flag, anti- Myc, anti- HA and anti- Tubulin monoclonal antibody were diluted at 1: 1,000 (Beyotime); and HRP- conjugated anti- rabbit IgG or anti- mouse IgG (Abbkine) at 1: 5,000.

Techniques: Expressing, Transfection, Plasmid Preparation, Staining, Infection

Journal: eLife

Article Title: An arms race between 5’ppp-RNA virus and its alternative recognition receptor MDA5 in RIG-I-lost teleost fish

doi: 10.7554/elife.94898

Figure Lengend Snippet: Figure 4. MDA5 recognizes 5’ppp-RNA in M. miiuy and G. gallus. (A) Schematic representation of 5’ppp-SCRV and dsRNA. The product of in vitro transcription runs as a single product degraded by RNase I. (B) Pull-down of biotinylated or non-biotinylated dsRNA and 5’ppp-SCRV by mmiMDA5. MKC cells were transfected with mmiMDA5-Flag plasmid, and the input and immunoprecipitated MDA5 proteins were analyzed by Western blot. (C) MKC cells were transfected with 5’ppp-SCRV, 5’OH-SCRV (5’ppp-SCRV dephosphorylated by CIAP), or 5’pppGG-SCRV (5’ppp-SCRV capped by m7G cap analog), then IRF3 dimerization was analyzed by native gel electrophoresis. (D) WT and si-MDA5 MKC cells were transfected with 5’ppp- SCRV and poly(I:C)-HMW, then IRF3 dimerization was analyzed by native gel electrophoresis. (E) The cytoplasmic fraction of MKC cells transfected with Flag-tagged mmiMDA5 was incubated with biotinylated 5’ppp-SCRV, 5’OH-SCRV, or 5’pppGG-SCRV. RNA-protein complexes were pulled down using streptavidin affinity beads. Input and pull-down samples were analyzed by SDS-PAGE and immunoblotting using anti-Flag antibody. (F) Purity of recombinant mmiMDA5 and mmiMDA5-△RD were determined by SDS-PAGE and immunoblotting using anti-His antibody. (G) EMSA of 5’ppp-SCRV with mmiMDA5. For binding competition, indicated non-biotinylated RNAs (50-fold molar excess over the probe) were included. The asterisk marks the binding bands between mmiMDA5 protein and different RNA probes. (H) EMSA of 5’ppp-SCRV with mmiMDA5 or mmiMDA5-△RD. The asterisk marks the binding bands between mmiMDA5 or mmiMDA5-△RD proteins and 5’ppp-SCRV RNA probes. (I) Schematic representation of 5’ppp-VSV. The product of in vitro transcription runs as a single product degraded by RNase I. (J) Pull-down of dsRNA and 5’ppp-VSV by ggaMDA5. DF-1 cells were transfected with ggaMDA5-Flag plasmids, and the input and immunoprecipitated MDA5 proteins were analyzed by Western blot. (K) DF-1 cells were transfected with 5’ppp-VSV, 5’OH-VSV (5’ppp-VSV dephosphorylated by CIAP), or 5’pppGG-VSV (5’ppp-VSV capped by m7G cap analog), then IRF3 dimerization was analyzed by native gel electrophoresis. (L) The cytoplasmic fraction of DF-1 cells transfected with Flag-tagged ggaMDA5 was incubated with biotinylated 5’ppp-VSA, 5’OH-VSV, or 5’pppGG-RNA. RNA-protein complexes were pulled down using streptavidin affinity beads. Input and pull-down samples were analyzed by SDS-PAGE and immunoblotting using anti-Flag antibody. (M) EMSA of 5’ppp-RNA with ggaMDA5. For binding competition, indicated non-biotinylated RNAs (50-fold molar excess over the probe) were included. The asterisk marks the binding bands between ggaMDA5 protein and different RNA probes.

Article Snippet: The antibody against MDA5 was diluted at 1: 500 (Beyotime, Cat# AF7164); The antibody against IRF3 was diluted at 1: 500 (Boster, Cat# BA4351- 2); anti- Flag, anti- Myc, anti- HA and anti- Tubulin monoclonal antibody were diluted at 1: 1,000 (Beyotime); and HRP- conjugated anti- rabbit IgG or anti- mouse IgG (Abbkine) at 1: 5,000.

Techniques: In Vitro, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Nucleic Acid Electrophoresis, Incubation, SDS Page, Recombinant, Binding Assay

Journal: eLife

Article Title: An arms race between 5’ppp-RNA virus and its alternative recognition receptor MDA5 in RIG-I-lost teleost fish

doi: 10.7554/elife.94898

Figure Lengend Snippet: Figure 5. Increased m6A modification and expression of MDA5 upon SCRV infection. (A) Schematic of the MeRIP-seq protocol used to identify differential m6A methylation following infection of M. miiuy spleen tissues with SCRV. (B) Circos plots showing differentially m6A-methylated peaks identified from normal and SCRV-infected spleen tissues of M. miiuy. The green and red lines on the innermost ring represented the m6A peaks identified from normal and SCRV-infected spleen tissues of M. miiuy, respectively. The orange lines represent the log2 fold change values for each differentially m6A peak. The blue spots represent the fold change of gene expression. Chromosomes were shown on the outermost ring, the ruler on which represented the physical distance is in millions of bases (Mb). (C) The m6A abundance in MDA5 mRNA transcripts detected by MeRIP-seq, the m6A peak of MDA5 is circled in the green box. (D) The exon1 sequence of MDA5 was submitted to the SRAMP website, and then the predicted m6A site was displayed. Four predicted methylation sites located on the exon1 of MDA5 were marked by red box, then we designed two m6A specific primers (MDA5-m6A-1 and MDA5-m6A-2). (E) m6A abundance on MDA5 exon1 detected by MeRIP-qPCR and emiquantitative PCR in MKC cells. (F) MeRIP-qPCR analysis of relative m6A level of MDA5 in Mock, SCRV-infected, and poly(I:C)-HMW-stimulated MKC cells. (G) Relative luciferase activity of pmirGLO- MDA5-exon1 firefly luciferase reporters in Mock and SCRV-infected (MOI = 5) MKC cells that treated with DMSO or Cycloleucine (CL, 20 mM). (H) mRNA levels of MDA5 in spleen and kidney tissues measured by qRT-PCR at indicated time after SCRV infection (MOI = 5). (I and J) mRNA and protein levels of MDA5 in MKC cells measured by qRT-PCR and Western blotting at indicated time after SCRV infection (MOI = 5) (I) and poly(I:C)-HMW stimulation (J). All data presented as the means ± SE from three independent triplicated experiments. **, p<0.01, as determined by Student’s t test (E-G) and one- way ANOVA with Dunnett’s multiple comparisons test (H-J).

Article Snippet: The antibody against MDA5 was diluted at 1: 500 (Beyotime, Cat# AF7164); The antibody against IRF3 was diluted at 1: 500 (Boster, Cat# BA4351- 2); anti- Flag, anti- Myc, anti- HA and anti- Tubulin monoclonal antibody were diluted at 1: 1,000 (Beyotime); and HRP- conjugated anti- rabbit IgG or anti- mouse IgG (Abbkine) at 1: 5,000.

Techniques: Modification, Expressing, Infection, Methylation, Gene Expression, Sequencing, Luciferase, Activity Assay, Quantitative RT-PCR, Western Blot

Journal: eLife

Article Title: An arms race between 5’ppp-RNA virus and its alternative recognition receptor MDA5 in RIG-I-lost teleost fish

doi: 10.7554/elife.94898

Figure Lengend Snippet: Figure 6. m6A-modification weakens MDA5 mRNA stability and antiviral ability. (A) Silence efficiency of si-METTL3 and si-METTL14 measured. Three siRNAs of METTL3 and METTL14 were transfected into MPC cells for 48 hr, respectively. (B) METTL3 immunoprecipitates with METTL14. EPC cells (1×107) were co-transfected with METTL3-Myc and METTL14-Flag expression plasmids for 24 hr, followed by immunoprecipitation (IP) with anti- Myc. (C) METTL3 and METTL14 significantly increased the m6A content. MPCs were transfected with si-Ctrl or si-METTL3 or si-METTL14 and MKCs were transfected with vector or METTL3 or METTL14 plasmids for 48 hr, then the m6A level was measured by colorimetry. (D) METTL3 and METTL14 overexpressed MKC cells seeded in 48-well plates overnight were treated with SCRV at the dose indicated for 48 hr. Then, cell monolayers were fixed with 4% paraformaldehyde and stained with 1% crystal violet. (E) MKC cells were transfected with pcDNA3.1 vector and METTL3 or METTL14 expression plasmid for 24 hr, then infected with SCRV (MOI = 5) for 24 hr. The qPCR analysis was conducted for SCRV-M and SCRV-G RNA levels. (F) The m6A level alteration of MDA5 upon METTL3 or METTL14 knockdown or overexpression was examined by MeRIP-qPCR. MPC cells were transfected with si-Ctrl or si-METT3 and MKC cells were transfected with vector or METTL3 or METTL14 plasmids for 48 hr. (G and H) MPC cells were transfected with si-Ctrl, si-METTL3 or METTL14 and MKC cells were transfected with vector, METTL3, or METTL14 plasmids for 48 hr, the expression of MDA5 was detected by qRT-PCR and western blotting. (I) MPC cells were transfected with si-Ctrl, si-METTL3, or si-METTL14, and MKC cells were transfected with vector, METTL3, or METTL14 plasmids, then 5 µg/ml actinomycin D was added to the cells for 0 hr, 2 hr, and 4 hr. The half-life of MDA5 was analyzed by qRT-PCR. (J) MKC cells were transfected with vector or METTL3&14 plasmids for 24 hr and then treated with Cycloleucine (CL) for 24 hr in a final concentration of 20 mM. The expression of MDA5 was detected by qRT-PCR and western blotting. (K) MKC cells were transfected with vector or METTL3&14 plasmids for 24 hr and then treated with CL for 24 hr at 20 mM, then 5 µg/ml actinomycin D was added to the cells for 0 hr, 2 hr, and 4 hr. The half-life of MDA5 was analyzed by qRT-PCR. (L) MKC cells were transfected with vector or METTL3&14 plasmids for 24 hr and stimulated with SCRV (MOI = 5) for 24 hr, then the expression of MDA5 was detected by qRT-PCR and western blotting. (M) MKC cells seeded in 48-well plates overnight were transfected with MDA5 or MDA5 + METTL3&14 plasmids for 48 hr, then the expression of MDA5 was detected by qRT-PCR. (N) MKC cells seeded in 48-well plates overnight were transfected with MDA5 or MDA5 + METTL3&14 plasmids were treated with SCRV at the dose indicated for 48 hr. Then, cell monolayers were fixed with 4% paraformaldehyde and stained with 1% crystal violet. (O) MKC cells were transfected with MDA5 or MDA5 +

Article Snippet: The antibody against MDA5 was diluted at 1: 500 (Beyotime, Cat# AF7164); The antibody against IRF3 was diluted at 1: 500 (Boster, Cat# BA4351- 2); anti- Flag, anti- Myc, anti- HA and anti- Tubulin monoclonal antibody were diluted at 1: 1,000 (Beyotime); and HRP- conjugated anti- rabbit IgG or anti- mouse IgG (Abbkine) at 1: 5,000.

Techniques: Modification, Transfection, Expressing, Immunoprecipitation, Plasmid Preparation, Colorimetric Assay, Staining, Infection, Knockdown, Over Expression, Quantitative RT-PCR, Western Blot, Concentration Assay

Journal: eLife

Article Title: An arms race between 5’ppp-RNA virus and its alternative recognition receptor MDA5 in RIG-I-lost teleost fish

doi: 10.7554/elife.94898

Figure Lengend Snippet: Figure 7. Detailed m6A regulatory mechanism of MDA5. (A) The binding relationship between YTHDF1, YTHDF2, or YTHDF3 and MDA5 mRNA was validated using a RIP assay. MKC cells were transfected with YTHDF1-Flag, YTHDF2-Flag, YTHDF3-Flag, or pcDNA3.1-Flag for 48 hr. (B) SiRNA silencing effect test of YTHDF1, YTHDF2, and YTHDF3. MPC cells were transfected with si-YTHDF1, si-YTHDF2, or si-YTHDF3 for 48 h. (C and D) MPC cells were transfected with si-YTHDF1, si-YTHDF2, si-YTHDF3 or si-Ctrl (C), and MKC cells were transfected with YTHDF1, YTHDF2, YTHDF3 or vector for 48 hr

Article Snippet: The antibody against MDA5 was diluted at 1: 500 (Beyotime, Cat# AF7164); The antibody against IRF3 was diluted at 1: 500 (Boster, Cat# BA4351- 2); anti- Flag, anti- Myc, anti- HA and anti- Tubulin monoclonal antibody were diluted at 1: 1,000 (Beyotime); and HRP- conjugated anti- rabbit IgG or anti- mouse IgG (Abbkine) at 1: 5,000.

Techniques: Binding Assay, Transfection, Plasmid Preparation