LPS-induced iNOS and nitric oxide in mouse splenic lymphocytes is enhanced by estrogen and regulated by miR-146a. An aliquot of 2.5 × 106 splenic lymphocytes (5 × 106/mL) from placebo- and estrogen-treated mice (n = 4 each) were stimulated with LPS or left unstimulated for 24 hours (medium only; A,B). Supernatants and cell pellets were collected for further analysis. (A) The production of nitric oxide in culture supernatants was determined with Griess assays. The graph shows means plus or minus SEM (n = 4 each). (B) Western blot analysis of the expression of iNOS in whole cell extracts. (C) The expression of iNOS mRNA in freshly isolated and 6 hours of LPS stimulated spenic lymphocytes was analyzed as indicated for Figure 1B. The graphs show the relative mRNA expression level with the means plus or minus SEM (n ≥ 3 each). (D) Freshly isolated splenic lymphocytes were transfected and stimulated with LPS as described for Figure 2B-D. The level of nitric oxide in supernatants from LPS-stimulated transfected cells was determined with Greiss assays. The graph shows means plus or minus SEM (n ≥ 6 each). (E) Western blot analysis of the expression of iNOS 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 iNOS signal detected by Western blotting 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 = 4). *P < .05; **P < .01.
