American Society of Hematology

Figure 6.

$PI3K inhibition by LY294002 treatment does not perturb FPN-GFP removal from phagosomes. (A) The effect of PI3K inhibition by LY294002 on the distribution of TfR-GFP and FPN-GFP is shown. RAW cells transfected with either TfR-GFP (top row) or FPN-GFP (bottom row) were pretreated with 100 μM LY294002 or with a vehicle control for 30 minutes. IgG-coated beads were then added to initiate phagocytosis, and cells were fixed 30 minutes after addition. Extracellular beads were labeled fluorescently (blue). Scale bars, 20 µm. (A) Arrowheads highlight the presence of TfR-GFP primarily at the plasma membrane in vehicle control–treated cells. Filled arrows emphasize enlarged TfR-GFP–containing endosomal vesicles, and open arrows indicate the perinuclear accumulation of TfR-GFP in LY294002-treated cells. The images acquired by widefield fluorescence microscopy are representative of at least 3 independent experiments. (B) The widefield fluorescent micrographs depict RAW cells expressing the PI(3)P-biosensor 2×FYVE-RFP (in red) and FPN-GFP (green) were pretreated with dimethyl sulfoxide (vehicle control) or 100 μM LY294002 (PI3K inhibitor) for 10 minutes. IgG-coated beads (1 μm in size) were added for 10 minutes to allow for phagocytosis prior to fixation. Beads that were not phagocytosed were marked with an AlexaFluor-647 secondary antibody prior to fixation and are colored blue. Arrowheads indicate FPN-negative phagosomes, whereas arrows indicate FPN-positive phagosomes. Scale bars, 20 µm. (C) The graph depicts the fraction of phagosomes positive for 2×FYVE-RFP or FPN-GFP from either vehicle control or LY294002-treated cells. The data represent the mean ± SEM from ≥100 phagosomes from 3 independent experiments. Statistical significance was determined using unpaired Student t tests with a Welch’s correction, where ***P < .001.$

PI3K inhibition by LY294002 treatment does not perturb FPN-GFP removal from phagosomes. (A) The effect of PI3K inhibition by LY294002 on the distribution of TfR-GFP and FPN-GFP is shown. RAW cells transfected with either TfR-GFP (top row) or FPN-GFP (bottom row) were pretreated with 100 μM LY294002 or with a vehicle control for 30 minutes. IgG-coated beads were then added to initiate phagocytosis, and cells were fixed 30 minutes after addition. Extracellular beads were labeled fluorescently (blue). Scale bars, 20 µm. (A) Arrowheads highlight the presence of TfR-GFP primarily at the plasma membrane in vehicle control–treated cells. Filled arrows emphasize enlarged TfR-GFP–containing endosomal vesicles, and open arrows indicate the perinuclear accumulation of TfR-GFP in LY294002-treated cells. The images acquired by widefield fluorescence microscopy are representative of at least 3 independent experiments. (B) The widefield fluorescent micrographs depict RAW cells expressing the PI(3)P-biosensor 2×FYVE-RFP (in red) and FPN-GFP (green) were pretreated with dimethyl sulfoxide (vehicle control) or 100 μM LY294002 (PI3K inhibitor) for 10 minutes. IgG-coated beads (1 μm in size) were added for 10 minutes to allow for phagocytosis prior to fixation. Beads that were not phagocytosed were marked with an AlexaFluor-647 secondary antibody prior to fixation and are colored blue. Arrowheads indicate FPN-negative phagosomes, whereas arrows indicate FPN-positive phagosomes. Scale bars, 20 µm. (C) The graph depicts the fraction of phagosomes positive for 2×FYVE-RFP or FPN-GFP from either vehicle control or LY294002-treated cells. The data represent the mean ± SEM from ≥100 phagosomes from 3 independent experiments. Statistical significance was determined using unpaired Student t tests with a Welch’s correction, where ***P < .001.

This Feature Is Available To Subscribers Only