Fig. 4

Nexturastat A enhances the phagocytic capacity of macrophages. A CD47 antibody titration (miap301) in SM1 cells by flow cytometry. Graph shows unblocked CD47 expression on melanoma cells at different concentrations of the anti-CD47 antibody. B Schematic representation of conditions used in phagocytosis assays. For figure panels C through K, BMDMs or THP-1-derived macrophages were unpolarized (M0) or polarized to M1-like or M2-like phenotype in the presence or absence of Nexturastat A (NextA, 5 µM). For figure panels C through J, melanoma cells were stained with CFSE and cocultured at a 2:1 ratio with macrophages, and phagocytosis was analyzed by flow cytometry. C Comparison of phagocytosis rates of M0, M1-like or M2-like BMDMs cocultured with CFSE stained SM1 cells. D-G Phagocytosis assays of untreated or NextA treated BMDMs cocultured with CFSE stained SM1 cells in the presence or absence of anti-CD47 or IgG isotype control (25 µg/ml). H-I Phagocytosis assays of BMDMs harvested from wild type C57BL/6 mice or HDAC6 knockout (KO) mice in the presence of anti-CD47 or isotype control. J Phagocytosis assays of THP-1-derived macrophages cocultured with WM164 human melanoma cells in the presence of human anti-CD47 or isotype control. All flow cytometry-based phagocytosis assays are quantified as % CFSE+ F4/80+ or CFSE+ CD11b+ cells out of total F4/80+ or CD11b+ cells. K Representative images of phagocytosis assays performed by confocal microscopy using BMDMs isolated from GFP mice (in green) and SM1 melanoma cells stained with CellTrace Far Red (shown in red). Nuclei were stained with DAPI (shown in blue). White arrow heads represent internalization of SM1 cells by macrophages. Scale bars represent 50 μm. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, non-significant