Fig 1.
TG6-44 treatment decreases A/X31-induced ROS in THP-1 cells.
A/X31-infected and PMA (50 μM)-treated THP-1 cells and PBMC incubated with TG6-44 (50μM), DPI (10 μM), or SOD (5 Units) were analyzed at 3 h and 6 h post-treatment for ROS levels, as described in Materials and Methods. Data represent results from one of three independent experiments. Values represent mean ± SEM.
Fig 2.
TG6-44 treatment leads to suppression of A/X31-induced inflammatory mediators in THP-1 cells.
Cell culture supernatants from A/X31-infected THP-1 cells treated with either vehicle and/or TG6-44 were harvested at 6 h (A) and 12 h (B) p.i. and assayed for inflammatory mediators by Bio-Plex assay, as described in Materials and Methods. Data represent results from four independent experiments. Values represent mean ± SEM.
Fig 3.
TG6-44 treatment leads to a decrease in NP-positive cells and altered expression patterns of NP in A/X31 infected THP-1 cells.
(A) Confocal fluorescence microscopy images evaluating localization patterns of NP are shown. Images were acquired from A/X31 infected THP-1 cells treated with vehicle and/or TG6-44 (50 μM) and harvested at 4 and 18 h p.i. Cell nuclei are shown as blue and NP as green. Scale bar corresponds to 10 μm. (B) Quantitative morphometric analysis showing the proportions of NP-positive cells in A/X31-infected THP-1 cells in the presence of vehicle or TG6-44. Values are the means ± S.D. of five 20x-image fields from one of two independent experiments. (C) Quantitative morphometric analysis showing the proportions of NP-positive cells expressing NP in the nucleus at 4 h p.i. with A/X-31, in the presence of TG6-44 and/or vehicle. Results are expressed as means ± S.D. (D) Quantitative morphometric analysis showing the proportions of NP-positive cells expressing NP in the cytoplasm at 18 h p.i. with A/X31 in the presence of TG6-44 and/or vehicle. Results are expressed as means ± S.D.
Fig 4.
TG6-44 treatment changes kinetics of virus infectivity in THP-1 cells.
(A) and (C); Cell lysates prepared from A/X31-infected cells treated with either vehicle or TG6-44 (50 μM) were analyzed for NP protein expression and β-actin, by western blot assay, at 6, 12, and 24 h p.i., as described in Materials and Methods. Data represent results from one of three independent experiments. (B) and (D); Cell culture supernatants from A/X31-infected cells treated with vehicle and/or TG6-44 (10 or 50 μm) were analyzed for virus titer at 24 h p.i by plaque assay, as described in Materials and Methods. Values in panel (B) and (D) represent mean ± SEM from three independent experiments.
Fig 5.
TG6-44 treatment decreases IFN-β levels in A/X31-infected THP-1 cells.
Cell cultures from untreated, mock-treated, and/or TG6-44-treated A/X31-infected THP-1 cells were analyzed for IFN-β mRNA in cell lysates (A) and IFN-β protein in cell culture supernatants (B) at 6, 12, and 24 h p.i., as described in Materials and Methods. A represents results from three independent experiments. B represents results from one of three independent experiments. Values represent mean ± SEM.
Fig 6.
TG6-44 treatment leads to decrease in A/X31-induced cell death in THP-1 cells.
A & B: A/X31-infected THP-1 cells treated with vehicle and/or TG6-44 were analyzed for percent annexin V+ and PI+ cells at 6, 12, and 24 h p.i. Representative FACS plots (A) and accumulative data from three independent experiments (B) are shown. Values in panel B represent percent mean ± SEM. C: Cell lysates from A/X31-infected THP-1 cells treated with vehicle and/or TG6-44 (50 μM) were assayed for Bcl-2, phosho Bcl-2, caspase-3, cleaved caspase-3, and β-actin, by western blot assay, as described in Materials and Methods. Data represent results from one of three independent experiments.