American Society of Hematology

Figure 7.

$Treatment of macrophages with NEM inhibits removal of FPN from the limiting phagosomal membrane. (A) RAW macrophages expressing FPN-GFP having phagocytosed biotinylated IgG-opsonized beads for 2 minutes prior to treatment with 2 mM NEM or vehicle control are shown. Extracellular beads (in magenta) were detected upon addition of NEM or vehicle by also adding AlexaFluor-647 conjugated avidin to the cells. Macrophages were fixed after 15 minutes, and the distribution of FPN-GFP around Alexa-647–negative phagosomes was analyzed. The hashed box demarcates the area of cells depicted in the insets. White arrows point to phagosomes that are FPN-GFP negative, whereas arrowheads point to sealed phagosomes (ie, that are avidin negative) that are FPN-GFP positive. Images were acquired using widefield fluorescence microscopy. Scale bars, 10 μm. (B) The graph depicts the fraction of FPN-GFP–positive phagosomes in the presence and absence of NEM 15 minutes after the addition of IgG targets. Note NEM was added 2 minutes after the addition of beads. The data are the mean ± SEM of 3 independent experiments. Statistical significance was determined by an unpaired Student t test, with a Welch’s correction; **P < .01. (C) The graph depicts the fraction of FPN-GFP–positive phagosomes in the presence of NEM at the indicated times on the x-axis. The data are the mean ± SEM of 3 independent experiments. Statistical significance was determined by 1-way analysis of variance with a Bonferroni multiple comparisons test.$

Treatment of macrophages with NEM inhibits removal of FPN from the limiting phagosomal membrane. (A) RAW macrophages expressing FPN-GFP having phagocytosed biotinylated IgG-opsonized beads for 2 minutes prior to treatment with 2 mM NEM or vehicle control are shown. Extracellular beads (in magenta) were detected upon addition of NEM or vehicle by also adding AlexaFluor-647 conjugated avidin to the cells. Macrophages were fixed after 15 minutes, and the distribution of FPN-GFP around Alexa-647–negative phagosomes was analyzed. The hashed box demarcates the area of cells depicted in the insets. White arrows point to phagosomes that are FPN-GFP negative, whereas arrowheads point to sealed phagosomes (ie, that are avidin negative) that are FPN-GFP positive. Images were acquired using widefield fluorescence microscopy. Scale bars, 10 μm. (B) The graph depicts the fraction of FPN-GFP–positive phagosomes in the presence and absence of NEM 15 minutes after the addition of IgG targets. Note NEM was added 2 minutes after the addition of beads. The data are the mean ± SEM of 3 independent experiments. Statistical significance was determined by an unpaired Student t test, with a Welch’s correction; **P < .01. (C) The graph depicts the fraction of FPN-GFP–positive phagosomes in the presence of NEM at the indicated times on the x-axis. The data are the mean ± SEM of 3 independent experiments. Statistical significance was determined by 1-way analysis of variance with a Bonferroni multiple comparisons test.

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