Inhibitors of P2X7 and NMMHC-IIA block phagocytosis. (A). Western blot images of NMMHC-IIA from iTHP-1 cell lysate immunoprecipitated by Dynabeads coated with P2X7 mAb. Cells pretreated with 1mM ATP show loss of coprecipitated NMMHC-IIA. Control cells were exposed to 1-μm YG beads (5 μL/mL) for the indicated time before wash and lysis. (B-C) Typical time-resolved flow cytometry dot plots showing phagocytosis of YG beads by human CD14+ monocytes and iTHP-1 cells. Cells were preincubated with inhibitors (CytD, 20μM for 20 minutes; KN-62, 1μM for 15 minutes; OxATP, 0.3mM for 30 minutes; S- and R-bleb, 100μM for 60 minutes; L4 mAb: 100 μg/mL) before the addition of beads. (D) Uptake of YG beads by iTHP-1 cells transfected with siRNA to P2RX7 or MYH9. All figures are representative of at least 3 experiments. (E) Inhibition of YG bead uptake by cells pretreated with L4 mAb or CytD before the addition of YG beads. Control values with human IgG (250 μg/mL) are shown. The arbitrary unit of area under YG bead uptake curve over 6.5 minutes is used to quantitate the phagocytosis level. *P < .05. n = 3 or 4.
