Fig. 5: SIRPα binding to HeLa cells and their phagocytosis by J774 macrophages are dependent on cholesterol content.

a–c Cholesterol depletion inhibits SIRPα binding and increases phagocytosis. a, b Cells were incubated with Cytochalasin D (CytD), Mn2 + , MβCD or anti CD47 antibodies (B6H12). Cells were next incubated with recombinant biotinylated human SIRPα Fc chimera protein, revealed by A647-streptavidin and imaged by confocal microscopy. a A Z stack of cells was acquired and the maximum projection is displayed with cells outlined in yellow. Scale bar, 10 µm. b Density of SIRPα binding on HeLa treated cells. Each dot corresponds to one cell coming from at least two independent experiments (Control: n = 106, anti-CD47: n = 60, Mn²⁺: n = 77, MβCD: n = 72 and CytD: n = 60). The mean and the standard deviation for each treatment are represented. *** denotes p < 0.0005, and n.s. denotes p > 0.05 as determined by a two-sided Student’s t test in comparison to the control. c Phagocytosis of HeLa treated cells by J774 macrophages. Harvested cells were treated as indicated in the Material and Methods section and washed before adding macrophages. Each point corresponds to an independent experiment (n = 6). The median for each treatment is represented.* denotes p < 0.05, and n.s. denotes p > 0.05 as determined by a two-sided Wilcoxon-Mann-Whitney’s test in comparison to the control.