Figure 3. Antagomir knock-down of miR-451 expression does not alter dendritic cell antigen presentation.

(A) FITC-labeled microRNA antagomirs were delivered to splenic dendritic cells by nucleofection and their uptake was assessed by flow cytometry after 24 h of culture (filled histogram) relative to mock-treated cells (unfilled histogram). Data are representative of 3 independent experiments. (B) miR-451 antagomir or control antagomir were delivered to splenic dendritic cells by nucleofection. RNA was isolated immediately following nucleofection, allowing insufficient time for the antagomir to interact specifically with target miRNA in order to assess whether the presence of antagomirs inhibit the qRT-PCR reaction (0 h). Cells were also cultured for 21 h prior to RNA isolation, which allowed for time for specific miRNA-antagomir interactions (21 h). Quantitative RT-PCR was performed and miR-451 expression levels are shown relative to sno202 expression levels. ND = not detected as below the detection limit of the assay. Data are representative of ≥ 3 independent experiments. (C) Cells were nucleofected with either control antagomir, antagomir specific for miR-685, or untreated, then cultured for 21 h. miR-451 expression levels were quantified by qRT-PCR as described in B, normalized to sno202 expression levels and shown relative to cells that did not undergo nucleofection. Means ± SEM are shown for 3 independent experiments. (D) Splenic dendritic cells from wild type or miR-451^null mice or wild type cells treated with antagomirs were infected with influenza for 24 h. Cell activation was assessed by staining for surface expression of MHCII or CD80 and overlay plots are shown for cells with normal (dark line) or reduced (light line) miR-451 expression. Data are representative of 3 independent experiments. (E) Splenic dendritic cells were isolated from WT or miR-451^null mice and wild type cells treated with miR-451 antagomir or control antagomir where indicated. Dendritic cells were infected with influenza, pulsed with ova peptide SIINFEKL, and cultured with CD8⁺ OT-1 T cells for 3 days. IFN-γ was measured in the supernatants of three biological replicates and the means ± SEM are shown. *p<0.05.