Journal: bioRxiv

Article Title: Epoxyeicosatrienoic acids and sEH inhibition prevent cardiac dysfunction in CVB3-induced myocarditis by positively regulating type I interferon signaling

doi: 10.1101/2023.02.03.527086

Figure Lengend Snippet: ( A ) Phosphorylation of IRF3 (on Ser396) in uninfected (CON) or CVB3-infected AC16 cells treated with 11,12-EET, 14,15-EET or TPPU (n=4). ( B ) Dimerization of IRF3 in uninfected (CON) or CVB3-infected AC16 cells treated with 11,12-EET, 14,15-EET or TPPU (n=4). ( C ) Nuclear translocation of IRF3 in uninfected (CON) or CVB3-infected AC16 cells treated with 11,12-EET, 14,15-EET or TPPU (n=4). GAPDH and LaminA/C were used as cytoplasm and nuclear protein loading controls. ( D ) Immunofluorescence images showing the impact of 14,15-EET and TPPU on the subcellular localization of IRF3 in uninfected (CON) or CVB3-infected AC16 cells; bars =50 μm. ( E ) Scheme showing the two main RNA sensor-induced IRF3 activation pathways. ( F-G ) AC16 cells that were uninfected (CON) or infected with CVB3 were treated with 11,12-EET, 14,15-EET or TPPU (n=3). Shown are changes in IRF3 phosphorylation following the siRNA-mediated down regulation of MDA5 (F), or TLR3 (G). ( H ) Activity of a IFNβ-luciferase reporter construct in HEK293 cells expressing either a control vector (vector), pcMDA5, pcMAVS, pcTBK1 , or pcIRF3-5D and treated with solvent (CON) or 14,15-EET (1 μM) for 24 hours. ( I ) Impact of the siRNA-mediated downregulation of TBK1 on the phosphorylation of IRF3 (on Ser396) in uninfected (CON) or CVB3-infected AC16 cells treated with 11,12-EET, 14,15-EET or TPPU (n=3). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: After 48 hours, transfected HEK293 cells were harvested in immunoprecipitation (IP) buffer containing 20 mM Tris (pH7.5), 150 mM NaCl, 1% Triton X-100 with freshly added PMSF, protease inhibitors and phosphatase inhibitors (Boster Biological Tech, Wuhan, China).

Techniques: Phospho-proteomics, Infection, Translocation Assay, Immunofluorescence, Activation Assay, Activity Assay, Luciferase, Construct, Expressing, Control, Plasmid Preparation, Solvent

Journal: bioRxiv

Article Title: Epoxyeicosatrienoic acids and sEH inhibition prevent cardiac dysfunction in CVB3-induced myocarditis by positively regulating type I interferon signaling

doi: 10.1101/2023.02.03.527086

Figure Lengend Snippet: ( A ) Impact of the siRNA-mediated downregulationof GSK3β on the phosphorylation of IRF3 (on Ser396) in uninfected (CON) or CVB3-infected AC16 cells treated with 11,12-EET, 14,15-EET or TPPU (n=4). ( B ) Impact of the EET antagonist; EEZE (1 μM) on the phosphorylation of GSK3β in AC16 cells treated with 11,12-EET, 14,15-EET or TPPU (n=4). ( C ) Co-immunoprecipitation of TBK1 with a GSK3β-Flag fusion protein from HEK293 cells treated with 14,15-EET (1 μM) or TPPU (10 μM) in the absence or presence of EEZE (n=3). ( D ) Impact of the wild-type GSK3β and the Y216F and Y216D GSK3β mutants on the phosphorylation of TBK1 in uninfected and CVB3-infected HEK293 cells (n=3). ( E-F ) Impact of GSK3β mutation alone and in combination with 14,15-EET on the phosphorylation of TBK1 in uninfected and CVB3-infected HEK293 cells (n=3-4). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: After 48 hours, transfected HEK293 cells were harvested in immunoprecipitation (IP) buffer containing 20 mM Tris (pH7.5), 150 mM NaCl, 1% Triton X-100 with freshly added PMSF, protease inhibitors and phosphatase inhibitors (Boster Biological Tech, Wuhan, China).

Techniques: Phospho-proteomics, Infection, Immunoprecipitation, Mutagenesis