2compared with THP-1 cells treated with shControl following treatment with poly I:C, poly dA:dT, STING ligands, c-di-AMP, and c-di-GMP or infection with HSV-1 or Sendai virus (Fig. selected positive regulator candidate genes, followed by infection with 10 MOI HSV-1. (mRNA in THP-1 cells transfected with nontargeting siRNA (siControl) or and mRNA in THP-1 cells treated with siControl or siBTN3A1, followed by incubation with LPS or extracellular poly I:C for 2 h. (and mRNA in MDMs silenced by siControl or siBTN3A1 using electroporation, followed by infection with HSV-1 (10 MOI) or SeV (1 MOI) for 4 h. Immunoblot and qRT-PCR analyses of the knockdown of endogenous in MDMs treated with siControl or siBTN3A1 were also performed. (isoforms, and mRNA in THP-1 cells treated with the indicated siRNA and then Valnoctamide stimulated with dsDNA for 6 h. * 0.05, ** 0.01, and *** 0.001 versus cells transfected with siControl (Students test). Data are representative of three independent experiments (mean SD). Table S1. siRNA library used in this study mRNA in THP-1 cells treated with control siRNA, siRNAs targeting 0.05 and ** 0.01 versus cells transfected with control siRNA (Students test). Data are representative of three independent experiments (mean SD). To characterize the physiological function of BTN3A1 in nucleic acid-induced responses, we used a knockdown strategy involving four distinct siRNAs targeting the human BTN3A1 gene. Differentiated THP-1 cells treated with these siRNAs exhibited a considerable reduction in BTN3A1 expression. Moreover, the mRNA expression level of induced by dsDNA was significantly decreased in had remarkable silencing effects without affecting cell viability (Fig. S1led to a substantial reduction in the production of and upon induction by cytosolic nucleic acids (Fig. 1did not inhibit the production of and when coupled with stimulation by the TLR ligands LPS and poly I:C (Fig. 1and were observed in knockdown MDMs, supporting the hypothesis that BTN3A1 acts as a positive regulator of type I IFN Valnoctamide responses induced by cytosolic DNA and RNA (Fig. 1mRNA and protein, revealing the specificity of BTN3A1 in type I IFN responses (Fig. 1was knocked down using lentiviral shRNA constructs. The reduced expression of was confirmed at the protein and mRNA levels (Fig. 2compared with THP-1 cells treated with shControl following treatment with poly I:C, poly dA:dT, STING ligands, c-di-AMP, and c-di-GMP or infection with HSV-1 or Sendai virus (Fig. 2suppressed cytoplasmic poly dA:dT-, c-di-AMPC, or c-di-GMPCinduced activation of activation, and IFN-stimulatory genes (Fig. S2 and (shBTN3A1). (in THP-1 cells treated with shControl or shBTN3A1 and then stimulated for 4 h with poly dA:dT after reconstitution with GFP or BTN3A1. (and mRNA in wild-type (WT) and knockout (KO) HeLa cells transfected with poly I:C or poly dA:dT or infected with SeV for 4 h. (in both WT and KO cells inoculated with SeV at the indicated time periods. N.D., not detected. (KO HeLa cells were stimulated for 3 h with poly I:C, poly dA:dT, or SeV. Cell lysates were analyzed by immunoblotting. (KO HeLa cells. The nuclei of the cells were stained with DAPI (blue). Original magnification, 40. (Scale bars, 20 Valnoctamide m.) Data are presented as the mean SD of three independent experiments. * 0.05, ** 0.01, and *** 0.001 (Students test). The band intensities of phosphorylated IRF3 (S386) were quantified by densitometry and are shown relative to the intensities of the corresponding total IRF3 bands. Open in a separate window Fig. S2. BTN3A1 is a positive regulator of type I IFN signaling. (and mRNA in THP-1 cells treated with either shControl or shBTN3A1, followed by transfection with low-molecular-weight (L.M.W.) poly I:C, high-molecular-weight (H.M.W.) poly I:C, poly dA:dT, c-di-AMP, or c-di-GMP or by infection with HSV-1, SeV, or RSV for 6 h. (mRNA in THP-1 cells treated with control shRNA or shRNA targeting the coding sequence (CDS) or 3 UTR of 0.05, ** 0.01, and *** 0.001 (Students test). During the nucleic acid-mediated immune response, the production of IFN- depends on the TBK1-induced phosphorylation DUSP1 of IRF3, followed by IRF3 dimerization and finally nuclear translocation of IRF3. The phosphorylation of S386 and S396 is critical for IRF3 dimerization and.