Journal: EMBO Reports

Article Title: Degradation of WTAP blocks antiviral responses by reducing the m 6 A levels of IRF3 and IFNAR1 mRNA

doi: 10.15252/embr.202052101

Figure Lengend Snippet: A, B Real‐time PCR (qRT–PCR) analyses of WTAP mRNA expression in PBMCs (A) or BMDMs (B) upon challenge with VSV‐eGFP, poly(I:C), HSV‐1, or HSV‐60 at indicated time points. n = 3 independent biological replicates, and error bars represent standard deviations. Data were compared using Student’s t ‐test. C qRT–PCR analyses of WTAP mRNA expression in A549 cells upon challenge with VSV‐eGFP, poly(I:C), HSV‐1, or poly (dA:dT) at the indicated time points. n = 3 independent biological replicates, and error bars represent standard deviations. Data were compared using Student’s t ‐test. D qRT–PCR analyses of WTAP mRNA expression in A549 cells stimulated with IFN‐α or IFN‐β at the indicated time points. n = 3 independent biological replicates, and error bars represent standard deviations. Data were compared using Student’s t ‐test. E A549 cells were stimulated with poly (I:C) for 16 h and then treated with PR‐171 (10 μM) or PS‐341 (10 μM) for additional 4 and 8 h. The cell lysates were analyzed by immunoblotting. Representative images from three independent biological experiments are shown (left), and the relative levels of WTAP are presented as the mean ± SD (right). F A549 cells were stimulated with poly (I:C) for 16 h and then treated with CQ (50 mM) or Baf A1 (0.2 mM) for additional 4 and 8 h. The cell lysates were analyzed by immunoblotting. LC3B served as a good marker of the autophagy process. Representative images from three independent biological experiments are shown (left), and the relative levels of WTAP are presented as the mean ± SD (right). G Immunoassays of extracts of A549 cells infected with VSV‐GFP for 16 h and then treated with PR‐171 (10 μM) or DMSO for additional 4 and 8 h, followed by immunoprecipitation with anti‐WTAP antibody and immunoblot analysis with anti‐Ub antibody. Data are representative of three independent biological experiments. H Coimmunoprecipitation and immunoblot analyses of extracts of 293T cells transfected with various combinations of plasmids encoding FLAG‐tagged WTAP, and HA‐tagged K48‐linked, K63‐linked or wild‐type ubiquitin and treated with DMSO or PR‐171 (10 μM). Data are representative of three independent biological experiments. Source data are available online for this figure.

Article Snippet: Anti‐RIG‐I, MDA5, WTAP, METTL14, MAVS, STING, Ub, STAT1, STAT2, LC3B, phosphorylated TBK1, phosphorylated IRF3, and phosphorylated STAT1 antibodies were purchased from Cell Signaling Technology; anti‐MxA, RSAD2, TBK1, MyD88, TRIF, and ICP‐27 antibodies were purchased from Abcam; anti‐WTAP, METTL3, IRF3, IFNAR1, IFNAR2, GAPDH, ISG15, HA, and Flag antibodies were purchased from Proteintech; anti‐IRF3 and MAVS antibodies were purchased from Santa Cruz, anti‐VSV‐G antibody was purchased from Sigma; anti‐SeV antibody was purchased from MBL.

Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Western Blot, Marker, Infection, Immunoprecipitation, Transfection