Myosin-II accumulation at the phagocytic synapse is strongly promoted by target rigidity and more weakly inhibited by CD47. (A) The phagocytic synapse schematic (left) illustrates the intensity analysis of immunofluorescence images. White arrow indicates alignment. THP-1 macrophages were either pretreated or not with myosin-II inhibitor blebbistatin (20 μM), followed by incubation with the various antiserum opsonized RBCs for 45 minutes at 37°C and then fixed and immunostained for myosin-IIA (green), F-actin (red), and DNA (blue) (scale bar, 10 μm). (B) Accumulation of myosin-II at the phagocytic synapse was quantified (n ≥ 3 ± SD), proving highest for rigid GA discocytes and secondarily for CD47-blocked RBCs (*P < .05). Blebbistatin suppresses myosin-IIA accumulation to levels similar to native RBCs, denoted CD47⁺ (**P < .05). (C) Fluorescence images of myosin-IIA localization in macrophage fed either GA discocytes with or without pretreatment with blebbistatin (20 μM), or CD47-blocked RBCs (scale bar, 30 μm). (D) Actomyosin fiber formation was quantified in macrophages, showing that addition of GA discocytes resulted in the highest frequency of macrophages with stress fibers. Blebbistatin pretreated macrophages showed curved and relaxed fibers, whereas macrophages cultured with native RBCs do not show fibers. (Inset) Immunoblot for nonmuscle myosin-IIA heavy chain, of macrophage lysates following phagocytosis, shows the presence of 230- and 520-kDa bands, with the high molecular weight band suggesting stable myosin assembly.