miR-223, but not miR-451 and miR-486, regulate LPS-induced IFNγ in splenic lymphocytes from estrogen-treated mice. Freshly isolated splenic lymphocytes (1.5 × 107) from estrogen-treated mice were transfected with either a negative inhibitor (control), miR-223, miR-451, or miR-486 inhibitors. Twenty-four hours after transfection, cells were stimulated with LPS for 24 hours, and the supernatants and cell pellets were collected for analysis. (A,B) The level of IFNγ and nitric oxide in culture supernatants of LPS-stimulated cells were determined by ELISAs (A) and Griess assays (B), respectively. The level of IFNγ and nitric oxide in supernatants from specific inhibitor transfected cells were presented as the percentage level of negative control inhibitor transfected cells. The graphs show the means plus or minus SEM (n = 5 each). (C) Western blot analysis of the expression of IFNγ protein in miRNA inhibitor transfected cells. Representative Western images are shown from at least 3 independent experiments. Densitometry analysis of IFNγ signal was performed as described for Figure 2. The graph shows relative density with means plus or minus SEM (n = 4 each). *P < .05.
