Figure S2.
A PI3K, Calcium, and NOX axis drive macrophage Nrf2 activation. (A and B) Expression of NoxRNAi decreased levels of ROS at the cell body in stage 15 macrophages (A and A′) and caused hypervacuolation (B). (C) Overexpression of Dp110D954A reduced levels of total cellular and phagosomal-localized PIP3 in stage 13 macrophages. (D) Overexpression of PI3KRNAi decreased levels of ROS at the cell body in stage 15 macrophages. (E) Domain organization of Drosophila Nox and human NOX5 as predicted using Interproscan (Blum et al., 2021). Both proteins have a highly conserved, calcium-sensitive EF-hand domain at the N-terminus as shown by sequence alignment. (F)srp > RyrRNAi reduced phagosomal ROS in stage 15 macrophages using RNAi construct TRiPHM05130. (G) Inhibitory concentrations of Ryanodine significantly decreased superoxide production within stage 13 macrophages. (H) Exposure of stage 15 embryos to 1 μM Thapsigargin (TG) induced the release of calcium in macrophages. (I) Thapsigargin did not rescue phagosomal ROS production by stage 15 H99 macrophages. (J–L) Reduced dNrf2 activation was observed upon NoxRNAi (J and J′), PI3KRNAi (K and K′) and overexpression of Dp110D954A (L and L′) in stage 15 macrophages. Cell bodies are indicated by dashed outline. Macrophages labeled in green (srp > GFP, A, D, G, and I) or red (srp > mchMoesin, H; fascin, J, K, and L). ROS production detected with DHE (in magenta, A, D, G, and I). dNrf2 activation evaluated using GstD1, ARE-GFP reporter (green, J, K, and L) *P < 0.05, ****P < 0.0001 via Mann–Whitney test (A′, B, C, D′, F, G′, H′, J′, K′, and L′). Images collected from: 26 control and 22 srp > NoxRNAi embryos; 24 control and 24 srp > PI3KRNAi embryos; 60 control and 57 srp > RyrRNAi embryos (F); 98 control and 51 Ryanodine-treated embryos (G); 12 DMSO only and 10 1μM TG embryos (H); 24 control and 19 H99 and 10 TG-treated H99 embryos (I); 10 control and 10 srp > NoxRNAi embryos (J); 7 control and 7 srp > PI3KRNAi embryos (K); 5 control and 7 srp > Dp110D954A embryos (L). Also see Fig. 3.

A PI3K, Calcium, and NOX axis drive macrophage Nrf2 activation. (A and B) Expression of NoxRNAi decreased levels of ROS at the cell body in stage 15 macrophages (A and A′) and caused hypervacuolation (B). (C) Overexpression of Dp110D954A reduced levels of total cellular and phagosomal-localized PIP3 in stage 13 macrophages. (D) Overexpression of PI3KRNAi decreased levels of ROS at the cell body in stage 15 macrophages. (E) Domain organization of Drosophila Nox and human NOX5 as predicted using Interproscan (Blum et al., 2021). Both proteins have a highly conserved, calcium-sensitive EF-hand domain at the N-terminus as shown by sequence alignment. (F)srp > RyrRNAi reduced phagosomal ROS in stage 15 macrophages using RNAi construct TRiPHM05130. (G) Inhibitory concentrations of Ryanodine significantly decreased superoxide production within stage 13 macrophages. (H) Exposure of stage 15 embryos to 1 μM Thapsigargin (TG) induced the release of calcium in macrophages. (I) Thapsigargin did not rescue phagosomal ROS production by stage 15 H99 macrophages. (J–L) Reduced dNrf2 activation was observed upon NoxRNAi (J and J′), PI3KRNAi (K and K′) and overexpression of Dp110D954A (L and L′) in stage 15 macrophages. Cell bodies are indicated by dashed outline. Macrophages labeled in green (srp > GFP, A, D, G, and I) or red (srp > mchMoesin, H; fascin, J, K, and L). ROS production detected with DHE (in magenta, A, D, G, and I). dNrf2 activation evaluated using GstD1, ARE-GFP reporter (green, J, K, and L) *P < 0.05, ****P < 0.0001 via Mann–Whitney test (A′, B, C, D′, F, G′, H′, J′, K′, and L′). Images collected from: 26 control and 22 srp > NoxRNAi embryos; 24 control and 24 srp > PI3KRNAi embryos; 60 control and 57 srp > RyrRNAi embryos (F); 98 control and 51 Ryanodine-treated embryos (G); 12 DMSO only and 10 1μM TG embryos (H); 24 control and 19 H99 and 10 TG-treated H99 embryos (I); 10 control and 10 srp > NoxRNAi embryos (J); 7 control and 7 srp > PI3KRNAi embryos (K); 5 control and 7 srp > Dp110D954A embryos (L). Also see Fig. 3.

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