Journal: Redox Biology

Article Title: Regulation of type I interferon responses by mitochondria-derived reactive oxygen species in plasmacytoid dendritic cells

doi: 10.1016/j.redox.2017.07.016

Figure Lengend Snippet: AMA effects on the production of type I IFNs and phosphorylation of IRF7 in TLR9-activated GEN2.2 cells. Cells were stimulated with the TLR9 agonist CpG-A (1 μM) in the presence or absence of AMA (0.5 μg/ml). After 6 h of stimulation the mRNA expression of IFNA1 was measured by real-time PCR (A). The level of secreted IFN-α cytokine was determined by ELISA (B). Cells were stimulated for 30 min as described above then the levels of the phosphorylated (p-IRF7) and native form of IRF7 were determined by Western blotting (C, D). Bars represent the means ± SD of three individual experiments (A, B, D) and a representative blot is shown (C). ***p < 0.001, ****p < 0.0001 vs. control, # p < 0.05, ## p < 0.01, #### p < 0.0001 vs. CpG-A. AMA: Antimycin-A, ND: not determined.

Article Snippet: For activation, cells were exposed to the TLR9 agonist CpG-A (ODN 2216, 1 μM; Cat. no. HC4037 from Hycult Biotech, Uden, The Netherlands) in the absence or presence of AMA (0.5 μg/ml; Sigma-Aldrich) for 6 h. The optimal concentration of AMA was chosen based on preliminary experiments performed on GEN2.2 cells.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Redox Biology

Article Title: Regulation of type I interferon responses by mitochondria-derived reactive oxygen species in plasmacytoid dendritic cells

doi: 10.1016/j.redox.2017.07.016

Figure Lengend Snippet: Dose- and time-dependent induction of cytosolic RIG-I receptor in GEN2.2 cells and the expression of RIG-I in AMA-exposed cells . In order to induce the cytosolic expression of RIG-I GEN2.2 cells were treated with increasing concentration of the specific TLR9 ligand, CpG-A (ranging from 0.01 to 0.5 μM). After 16 h the presence of RIG-I was detected in the cell lysates by Western blotting (A, B). To evaluate the time-dependent induction of RIG-I, GEN2.2 cells were exposed to 0.25 μM of CpG-A then the expression of RIG-I was measured in different time points (C, D). Finally cells were exposed to 0.25 μM of CpG-A in the presence or absence of AMA (0.5 μg/ml) or left untreated, for 16 h. Then cells were lysed and the protein level of RIG-I was assessed (E, F). Representative blots (A, C, E) and the means ± SD of four independent experiments (B, D, F) are shown. ***p < 0.001, ****p < 0.0001 vs. controls, ### p < 0.001 vs. CpG-A. AMA: Antimycin-A, ND: not determined.

Article Snippet: For activation, cells were exposed to the TLR9 agonist CpG-A (ODN 2216, 1 μM; Cat. no. HC4037 from Hycult Biotech, Uden, The Netherlands) in the absence or presence of AMA (0.5 μg/ml; Sigma-Aldrich) for 6 h. The optimal concentration of AMA was chosen based on preliminary experiments performed on GEN2.2 cells.

Techniques: Expressing, Concentration Assay, Western Blot

Journal: Redox Biology

Article Title: Regulation of type I interferon responses by mitochondria-derived reactive oxygen species in plasmacytoid dendritic cells

doi: 10.1016/j.redox.2017.07.016

Figure Lengend Snippet: RIG-I and TLR9 induced type I IFN production in CpG-A-pre-conditioned GEN2.2 cells in the presence of AMA. Cells were pre-treated with CpG-A (0.25 μM) for 16 h to induce the cytosolic expression of RIG-I. Following thorough washing steps cells were re-exposed to the specific RIG-I ligand 5′ppp-dsRNA (RIGL, 1 μg/ml) in the presence or absence of AMA (0.5 μg/ml). The IFNA1 mRNA expression level was determined by real-time PCR after 3 h (A) and IFN-α protein level was assessed by ELISA after 6 h (B). In parallel experiments the re-activation of the cells was carried out with high dose of CpG-A (1 μM) in combination or not with AMA (0.5 μg/ml). IFNA1 mRNA (C) and IFN-α protein levels (D) were measured as in (A) and (B), respectively. Data are presented as means ± SD of four individual experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. control, # p < 0.05 vs. RIGL and ## p < 0.01 vs. CpG-A. AMA: Antimycin-A, ND: not determined.

Article Snippet: For activation, cells were exposed to the TLR9 agonist CpG-A (ODN 2216, 1 μM; Cat. no. HC4037 from Hycult Biotech, Uden, The Netherlands) in the absence or presence of AMA (0.5 μg/ml; Sigma-Aldrich) for 6 h. The optimal concentration of AMA was chosen based on preliminary experiments performed on GEN2.2 cells.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Activation Assay

Journal: Redox Biology

Article Title: Regulation of type I interferon responses by mitochondria-derived reactive oxygen species in plasmacytoid dendritic cells

doi: 10.1016/j.redox.2017.07.016

Figure Lengend Snippet: Effects of AMA treatments on the first and second waves of type I IFN production in primary human pDCs. Freshly isolated primary pDCs were stimulated with the TLR9 agonist CpG-A (1 μM) in the presence or absence of AMA (0.5 μg/ml). After 6 h of stimulation the mRNA expression of IFNA1 was measured by real-time PCR (A) and the level of secreted IFN-α cytokine was determined by ELISA (B). To investigate the effects of AMA on the second wave of type I IFN responses, primary pDCs were pre-treated with CpG-A for 16 h then after thorough washing steps cells were re-exposed to 5′ppp-dsRNA (RIGL, 1 μg/ml) in the presence or absence of AMA (0.5 μg/ml). After 3 h the IFNA1 mRNA expression level was determined by real-time PCR (C) and after 6 h IFN-α protein level was assessed by ELISA (D). Data are presented as means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. control, # p < 0.05 vs. CpG-A or RIGL and ## p < 0.01 vs. CpG-A or RIGL. AMA: Antimycin-A, ND: not determined.

Article Snippet: For activation, cells were exposed to the TLR9 agonist CpG-A (ODN 2216, 1 μM; Cat. no. HC4037 from Hycult Biotech, Uden, The Netherlands) in the absence or presence of AMA (0.5 μg/ml; Sigma-Aldrich) for 6 h. The optimal concentration of AMA was chosen based on preliminary experiments performed on GEN2.2 cells.

Techniques: Isolation, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay