Nef treatment of microglial cells induces iNOS expression.

<p>BV-2 cells (<b>A</b>) or purified primary murine microglial cells (<b>B</b>) were treated for the indicated time with myr⁺Nef_SF2 (100 ng/ml in <b>A</b>, 200 ng/ml in <b>B</b>, closed circles). Total cellular RNAs were isolated and real time RT-PCR analysis was performed as reported in the materials and methods section. Results were expressed using the 2^-ΔΔCT method using basal mRNA level in untreated cells (Ctr, open circles) at T = 0 as a calibrator and GAPDH level as an internal loading control. (<b>C</b>, <b>D</b>) BV-2 cells were treated for 24 h with the indicated amounts of myr⁺Nef_SF2, heat inactivated myr⁺Nef_SF2 (Inactiv. recNef), LPS or heat treated LPS. Total cell lysates were analyzed by Western Blot for iNOS (<b>C</b>, upper panel) levels and, as internal loading control, β-Tubulin expression (<b>C</b>, lower panel). (<b>D</b>) NO₂^- content in the supernatants was quantified using the Griess colorimetric assay as reported in the materials and methods section. Dark gray bars: native myr⁺Nef or LPS. Light gray bars: heat pre-treated (Inactiv.) myr⁺Nef or heat pre-treated LPS. Results from one of five independent experiments are shown. (<b>E</b>) Cell phenotyping by flow cytometry of human monocytes and M1 macrophages obtained as described in materials and methods. According to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130189#pone.0130189.ref077" target="_blank">77</a>], human monocytes were CD14⁺/CD163⁺/CD86⁺ whereas M1 macrophages were CD14^-/CD163^-/CD86^bright. (<b>F</b>) M1 human macrophages were left untreated or treated for 24, 48 and 72 h with IFNγ, 100 ng/ml wild type myr⁺Nef_SF2 plus IFNγ, heat pre-treated myr⁺Nef_SF2 and IFNγ or 100 ng/ml LPS plus IFNγ. Total cellular extracts were analyzed by Western Blot to evaluate iNOS expression using specific antibodies as described in materials and methods. β-tubulin expression as internal loading control. Blots are representative of two independent experiments.