Figure 1.
Corpse uptake drives an oxidative burst and downstream Nrf2 activation in Drosophila macrophages. (A and B) ROS production in stage 15 macrophage, revealed by DHE (magenta, A) and H2DCFDA (green, B). (C and D) ROS levels (DHE) increased upon apoptotic corpse uptake. In C, solid and dashed outlines indicate naïve and primed macrophages in stage 12 embryos, respectively. In D, levels of superoxide at the macrophage cell body are plotted against the number of cytoplasmic vacuoles. (E) ROS release (white arrow) detected at nascent phagosomes (white asterisks) in stage 13 embryos. (F and G) Decreased ROS levels in stage 15 H99 macrophages, as detected by DHE (F) and H2DCFDA (G). (H and I) Stage 15 embryonic macrophages activated ARE-GFP reporters of Nrf2 activation (green) including the GstD1, ARE-GFP reporter (H′), or a synthetic ARE-GFP reporter for Nrf2 activity (I). (J) Decreased Nrf2 activation in stage 15 H99 macrophages using GstD1, ARE-GFP reporter. (K) FACS plots showing the gating strategy adopted on cell suspension from non-transgenic w1118Drosophila embryos to eliminate background fluorescence. Stage 15 H99 macrophages showed reduced activation of the GstD1, ARE-GFP reporter. (L) Relative GFP mRNA levels from sorted stage 15 macrophages of the indicated genotypes; data from two independent biological replicates. In all figures, the cell body is indicated by a dashed or solid white outline. Macrophages labeled in red (fascin, H, J; srp > mchMoesin, B, G) or green (srp > GFP, A, C, E, and F). ROS detected with DHE (magenta, A, C, E, and F) or H2DCFDA (green, G). ns: not significant, **P < 0.01, ***P < 0.001, ****P < 0.0001 via Mann–Whitney test (A′, A″, B′, C′, E′, F′, G′, H′, I, J′) or one-way ANOVA followed by Dunnett’s multiple comparison analysis (K″, L). Images collected from 7 uninjected and 5 injected embryos (A); 3 uninjected and 7 embryos (B); 8 embryos (C); 14 control and 18 H99 embryos (F); 26 control and 10 H99 embryos (G); 17 control and 10 GstD1, ARE-GFP embryos (H); 12 control and 10 H99 embryos (J).

Corpse uptake drives an oxidative burst and downstream Nrf2 activation in Drosophila macrophages. (A and B) ROS production in stage 15 macrophage, revealed by DHE (magenta, A) and H2DCFDA (green, B). (C and D) ROS levels (DHE) increased upon apoptotic corpse uptake. In C, solid and dashed outlines indicate naïve and primed macrophages in stage 12 embryos, respectively. In D, levels of superoxide at the macrophage cell body are plotted against the number of cytoplasmic vacuoles. (E) ROS release (white arrow) detected at nascent phagosomes (white asterisks) in stage 13 embryos. (F and G) Decreased ROS levels in stage 15 H99 macrophages, as detected by DHE (F) and H2DCFDA (G). (H and I) Stage 15 embryonic macrophages activated ARE-GFP reporters of Nrf2 activation (green) including the GstD1, ARE-GFP reporter (H′), or a synthetic ARE-GFP reporter for Nrf2 activity (I). (J) Decreased Nrf2 activation in stage 15 H99 macrophages using GstD1, ARE-GFP reporter. (K) FACS plots showing the gating strategy adopted on cell suspension from non-transgenic w1118Drosophila embryos to eliminate background fluorescence. Stage 15 H99 macrophages showed reduced activation of the GstD1, ARE-GFP reporter. (L) Relative GFP mRNA levels from sorted stage 15 macrophages of the indicated genotypes; data from two independent biological replicates. In all figures, the cell body is indicated by a dashed or solid white outline. Macrophages labeled in red (fascin, H, J; srp > mchMoesin, B, G) or green (srp > GFP, A, C, E, and F). ROS detected with DHE (magenta, A, C, E, and F) or H2DCFDA (green, G). ns: not significant, **P < 0.01, ***P < 0.001, ****P < 0.0001 via Mann–Whitney test (A′, A″, B′, C′, E′, F′, G′, H′, I, J′) or one-way ANOVA followed by Dunnett’s multiple comparison analysis (K″, L). Images collected from 7 uninjected and 5 injected embryos (A); 3 uninjected and 7 embryos (B); 8 embryos (C); 14 control and 18 H99 embryos (F); 26 control and 10 H99 embryos (G); 17 control and 10 GstD1, ARE-GFP embryos (H); 12 control and 10 H99 embryos (J).

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