Figure 6.
Avrainvillamide and BFA induce differentiation, increase phagocytosis and oxidative burst of OCI-AML3 cells. (a) OCI-AML3 cells were treated for 72 h with DMSO (CTR), AVA (0.5 μM), BFA (0.5 μM) or KPT-330 (0.1 μM) before live cells were stained and analyzed by flow cytometry for CD163, CD86, CD14 and CD11b expression by gating on living cells. Fold changes (mean±S.D.) relative to controls are shown (n=3). Comparisons between treated and control samples were conducted using an unpaired Student's t-test (***P<0.001). (b) OCI-AML3 cells were treated for 72 h with DMSO (CTR), avrainvillamide (AVA; 0.5 μM), BFA (0.5 μM) or KPT-330 (0.1 μM) and analyzed by flow cytometry and cytospun and stained with May-Grunwald-Giemsa before analyzed by microscopy (Zeiss axio Observer A1) using a × 40 objective lens. A representative histogram for AVA-treated cells is shown to the left and representative May-Grunwald-Giemsa stained images are shown (n=3). Scale bars, 10 μm. (c) OCI-AML3 cells treated with DMSO (CTR), AVA (0.5 μM), BFA or KPT-330 for 72 h followed by phagocytosis assay with opsonization of fluorescent bacteria. Quantification of increase in phagocytotic cells compared with CTR (n=3), results represent mean±S.D. (***P<0.001). (d) Localization of fluorescently labeled bacteria in 72 h BFA-treated OCI-AML3 cells stained with DAPI. Images were captured using Zeiss Axio ObserverZ1, × 63 oil objective. Scale bars, 10 μm. (e) OCI-AML3 cells were treated with DMSO (CTR), AVA (0.5 μM), BFA (0.5 μM) or KPT-330 (0.1 μM) for 72 h before 2 × 105 cells were incubated with nitroblue tetrazolium (NTB—reaction with ROS produces a dark blue color) and stimulated with PMA for 30 min at 37 °C. Cells were cytospun and counterstained with Safranin O and images were captured using Zeiss Axio Observer A1, × 10 objective (n=3). Scale bars, 50 μm. (f) BFA-treated cells from (e) were stained with additional DAPI. Images were captured using Zeiss Axio Observer A1, × 40 objective. Scale bars, 20 μm