LPS-induced IFNγ in mouse splenic lymphocytes is regulated by the estrogen-regulated miRNA, miR-146a. (A) Total RNA was isolated from freshly isolated splenic lymphocytes using mirVana miRNA isolation kits. Relative expression levels of miR-146a and miR-146b between splenic lymphocytes from placebo- and estrogen-treated mice were analyzed using the Taqman miRNA assay system. The graph shows the means plus or minus SEM (n ≥ 6 each). (B-D) Freshly isolated splenic lymphocytes (1.5 × 10⁷) from placebo- and estrogen-treated mice were transfected with either a negative mimic (control) or miR-146a mimic. Twenty-four hours after transfection, cells were left unstimulated (B) or stimulated with LPS for 24 hours (C,D), and then the supernatants and cell pellets were collected for analysis. (B) Western blot analysis of the expression of IRAK-1 in unstimulated cells at 48 hours after transfection. (C) The level of IFNγ in supernatants from LPS-stimulated miR-146a mimic transfected cells is shown as the percentage expression of negative control mimic transfected cells. Graph shows mean plus or minus SEM (n ≥ 6 each). (D) Western blot analysis of the expression of IFNγ in cell extracts from negative mimic and miR-146a mimic transfected cells stimulated with LPS. Representative Western blot images are shown from at least 3 independent experiments. Densitometry analysis of the IFNγ signal in blot images was performed using Kodak molecular imaging software (version 4.5) and normalized to the loading control β-actin. The graph shows relative density with means plus or minus SEM (n = 3 each). *P < .05; ***P < .001.