Figure 7

Expression and susceptibility to IFN stimulation of 90K in primary HIV-1 target cells. (A) cDNA was prepared from total RNA of the indicated cell lines and primary cells, and analyzed for relative 90K mRNA expression by TaqMan-based quantitative PCR with RNaseP expression as a reference. Depicted is the relative level of 90K expression, and values obtained for 293T cells were arbitrarily set to 1. Histograms represent arithmetic means ± S.E.M. of three independent experiments, except for the data for CD4⁺ T-cells, which shows arithmetic means of two experiments. (B) Indicated primary cell cultures were treated with IFN-α (100 U/ml) or IFN-γ (100 U/ml) for 24-48 hours, or left untreated, prior to RNA isolation and cDNA synthesis. Shown is the fold increase of 90K mRNA level from 2-3 experiments, each performed in duplicates. (C) Western Blot analysis of indicated cells treated with IFN-α (100 U/ml) or IFN-γ (100 U/ml) for 48 hours, or left untreated. (D) Anti-90K ELISA of cell lysates, following treatment with IFN-α (100 U/ml) or IFN-γ (100 U/ml) for 48 hours where indicated. (E) Immunofluorescence stain of indicated primary cell cultures. Cells treated with IFN-α (100 U/ml) or IFN-γ (100 U/ml) for 24-48 hours where indicated or left untreated. Scale bar: 10 μm. (F) Quantification of 90K immunofluorescence by KNIME image processing plug-in and the mean fluorescence intensity (MFI) signal per cell was determined. The data represent the arithmetic mean ± S.E.M. of 154 untreated, 259 IFN-α-treated and 147 IFN-γ-treated T-cells and the mean of 85 untreated, 121 IFN-α-treated and 64 IFN-γ-treated macrophages.