Extended Data Fig. 6: DNGR signalling promotes phagosomal ROS production.
a–b, Confocal images of RAW264.7 cells transfected with empty vector or plasmid encoding C9::C7 or C9(Y7F)::C7 receptors and pulsed with zymosan (a) or dead sRBCs (b) in the presence of Nitroblue tetrazolium (NBT) (Scale bar 10 μm). Quantification of ROS+ phagosomes. Data represented as mean (± s.e.m.) (a) or (± s.d.) (b) and are representative of two independent determinations (n = 2). P values determined by one-way ANOVA. c, RAW264.7 stably expressing C9 or C9(Y7F) receptors were pulsed with CellTracker DeepRed (CTDR)-labelled FP-sRBCs for 2 hrs. Percentage of CTDR+ RAW264.7 cells was quantified by flow cytometry. Data represented as mean (± s.d.) and are representative of two independent experiments (n = 2). d, Confocal images of RAW264.7 stably expressing C9 or C9(Y7F) receptors pulsed with dead cells in the presence of NBT for 2 hrs (scale bars 10 μm). Image is a representative image of three similar images. e, RT-PCR of NADPH oxidase subunits in HEK293T. Representative of two experiments (n = 2). f, HEK293T cells stably expressing C9::C7 were challenged with zymosan-Oxyburst in the presence or absence of DPI for 1 hr. Oxyburst+ positive phagosomes were quantified across 5 fields of view (n > 100 phagosomes). Data represented as mean (± s.e.m.). P values were calculated by unpaired parametric test, Mann-Whitney and are representative of two independent experiments (n = 2). g, HEK293T C9::C7 cells were pulsed with zymosan-Ova (left) or transfected with plasmid encoding VENUS-SIINFEKL (right) in the presence or absence of DPI (10 μM) for 4 hrs before fixing and adding of OT-I Rag1-/- T-cells. IFN-γ was assessed by ELISA, plotted as mean (± s.d.) of an experimental triplicate. n.s., not significant; *P ≤ 0.05; **P ≤ 0.01.
