1. TNF-α inhibits CMV MIEP activity in human astrocytes.
TNF-α inhibits CMV MIEP activity in human astrocytes. (A) Schematic description of various truncated MIEP LacZ reporter constructs used to test the effect of cytokines on promoter activity in human astrocytes. M, modulator sequence; U, unique region; E, enhancer sequence; P, basal promoter sequence; L, leader sequence). Numerical positions were assigned relative to the most distal region of the full-length MIEP used. (B to D) Primary human astrocytes, either treated with TNF-α for 48 h or untreated, were transfected with each of the indicated plasmids using Fugene 6 reagent (Roche, Indianapolis, IN). Culture lysates were harvested at 72 h posttransfection and analyzed for β-galactosidase activity, which was normalized to total protein and compared to expression from untreated cells. (B) β-Galactosidase expression from truncated MIEP constructs are expressed as units of optical density at 595 nm (OD595) per mg protein. (C and D) The effect of gross deletions of the MIEP on TNF-α-mediated suppression of promoter activity in astrocytes is expressed as percent suppression, where expression levels from TNF-α-treated astrocytes were compared to the expression of the same plasmid construct in untreated cells. Based on the pattern of reduction of reporter gene expression among various constructs, the effect of TNF-α is mediated largely at the level of the enhancer. Data are representative of at least three experiments performed with astrocytes from different donors.
Maxim C.-J. Cheeran et al. Clin. Microbiol. Rev. 2009; doi: /CMR