Lpd and VASP coordinate actin polymerization at the phagocytic cup. (A) Representative confocal projections of RAW 264.7 macrophages expressing GFP-Lpd (left) and GFP-Lpd^EVmut (right) after a 10 min incubation with IgG-opsonized SRBCs. Inside-outside staining was performed to differentiate between internalized/total (red) and non-internalized (blue) SRBCs. (B) Quantification of the phagocytic efficiency in RAW 264.7 macrophages overexpressing either Lpd^WT or Lpd^EVmut. Unpaired t test of individual phagocytic efficiency values n = 86 (Lpd^WT), n = 80 (Lpd^EVmut), across N = 3 independent experiments; data are means ± SEM. (C) Representative 3D reconstruction of RAW 264.7 macrophages expressing the Lpd^EVmut construct (green) during phagocytosis of IgG-opsonized SRBCs and stained with phalloidin (red). (i) Magnification of the area denoted by the dotted box (i) in main panel, depicting SRBCs (blue). (ii) Magnification of the area denoted by the dotted box (ii) in main panel, depicting a representative phagocytic cup from RAW 264.7 macrophages expressing either the Lpd^EVmut (left) or the Lpd^WT construct (right) for comparison. Similar observations were made in three independent experiments. Scale bars: 5 μm. (D) Representative confocal slice of RAW 264.7 macrophages co-expressing the Lpd^EVmut construct (green) and the cPHx3 construct (red) during phagocytosis. Insets represent the area denoted by the dotted box (left) and compared to the Lpd^WT-expressing macrophages (right). ****, P < 0.0001.