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