Nox-dependent release of ROS is regulated by PI3K and calcium. (A) RT-qPCR data from sorted stage 15 control and H99 macrophages (two independent biological replicates). (B and C)srp > Nox^RNAi decreased phagosomal ROS (B) and caused hypervacuolation (C). (D and D′) Macrophage PIP₃ (srp > GPH, white arrowhead) accumulated on nascent phagosomes (yellow asterisk) during embryonic stage 13. The image is a still from a movie of macrophage dispersal during stage 13 of development (see Video 1). (E and E′) Phagosomal ROS levels (DHE, magenta) in srp > Dp110^D954A macrophages. (F) Macrophage-specific inhibition of PI3K (srp > Dp110^D954A) caused hypervacuolation. (G and H) Inhibition of calcium release by srp > Ryr^RNAi reduced phagosomal ROS (G and G′ DHE, magenta) and Nrf2 activation (H and H′, GstD1, ARE-GFP reporter). (I) Schematic showing corpse uptake promotes PIP₃ and calcium-dependent Nox activation, driving ROS release and activation of Nrf2-mediated macrophage cytoprotection. In all figures, with the exception of D, images are representative of stage 15 macrophages. In all figures, the cell body is indicated by a white dashed outline. Macrophages labeled in red (anti-fascin, H) or green (B, C, G srp > GFP; D, E srp > GPH). ns: not significant, **P < 0.01, ***P < 0.001, ****P < 0.0001 via Mann–Whitney test (B′, C, D′, E′, F, G′, H′) or unpaired t test (A). Images collected from 26 control and 26 srp > Nox^RNAi embryos (B); 51 control, 33 srp > Duox^RNAi, and 27 srp > Nox^RNAi embryos (C); 20 control and 35 srp > Dp110^D954A embryos (E); 57 control and 68 srp > Ryr^RNAi embryos (G); 7 control and 11 srp > Ryr^RNAi embryos (H).