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. 2018 Jun 11;9:1327. doi: 10.3389/fimmu.2018.01327

Figure 6.

Figure 6

Involvement of Danio rerio DDX41 (DrDDX41) in nuclear factor-κB (NF-κB) signaling pathway. (A) Activation of the NF-κB-binding promoter in HEK293T cells transfected with an NF-κB luciferase reporter (NF-κB-Luc; 150 ng/mL), a renilla luciferase reporter (15 ng/mL) plus increasing amounts (0, 200, and 400 ng/mL) of expression vectors for DrDDX41 or Danio rerio STING (DrSTING) individually or expression vector for DrSTING (200 ng/mL) together with increasing amounts (0, 200, and 400 ng/mL) of expression vectors for DrDDX41 and stimulated with poly(dA:dT) (1 µg/mL) for 6 h. Data are the average luciferase activity ± SD (*p < 0.05; **p < 0.01). ns, not significant. (B) Western blot analysis for the expression of DrDDX41 and DrSTING in HEK293T cells transfected with increasing amounts (0, 200, and 400 ng/mL) of corresponding vectors. (C) DrDDX41 interacts with DrSTING. HEK293T cells were transfected with Flag-DrDDX41 and Myc-DrSTING for 24 h. Cell lysates were immunoprecipitated with anti-Myc antibody (Myc) or control mouse IgG (IgG), and analyzed by western blot using anti-Flag and anti-Myc antibodies. Expression of the transfected plasmids was analyzed with anti-Flag and anti-Myc antibodies in the whole cell lysates. (D) Activation of the NF-κB-binding promoters in embryos microinjected with an NF-κB luciferase reporter (NF-κB-Luc; 100 pg/embryo), a renilla luciferase reporter (10 pg/embryo), and poly(dA:dT) (100 pg/embryo) plus increasing amounts (0, 50, 100, and 200 pg/embryo) of expression vectors for DrDDX41 or DrSTING individually or expression vector for DrSTING (50 pg/embryo) together with increasing amounts (0, 50, 100, and 200 pg/embryo) of expression vectors for DrDDX41. Data are the average luciferase activity ± SD (*p < 0.05; **p < 0.01). (E) Activation of the NF-κB-binding promoter in embryos microinjected with an NF-κB luciferase reporter (NF-κB-Luc; 100 pg/embryo), a renilla luciferase reporter (10 pg/embryo) plus standard MO and PBS (US, unstimulated, administered with standard MO and PBS) or plus standard MO [Ctrl, administered with standard MO and poly(dA:dT)], DrDDX41/DrSTING-MO, the targeted-MOs together with their mRNAs, and poly(dA:dT) (100 pg/embryo). Data are the average luciferase activity ± SD (*p < 0.05; **p < 0.01). (F,G) Quantitative RT-PCR analysis of Danio rerio IL-6 (F) and Danio rerio TNFα (G) mRNA levels in embryos microinjected with standard MO and PBS (US) or plus standard MO (Ctrl), DrDDX41/DrSTING-MO, the targeted-MOs together with their mRNAs, and poly(dA:dT) (100 pg/embryo). Each of the MOs and mRNAs was administered at 4 ng/embryo and 200 pg/embryo in each group. Data are representative of three independent experiments as mean ± SD (*p < 0.05; **p < 0.01). Standard loading was indicated by β-actin expression.