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Fig. 3 NADPH oxidase is required for the Lc3-mediated response to S. aureus infection. (A) Confocal photomicrographs are shown as maximum intensity projections of the Lc3-mediated response in control (top panel) and cyba knockdown (bottom panel) of fixed CMV:GFP-Lc3 embryos infected with approximately 1500 CFU of mCherry-labeled S. aureus. Embryos were fixed at 1 hpi and chemically stained for Mpx activity (TSA, magenta). Scale bars: 10 µm. (B) Quantification of Lc3 associations with intracellular S. aureus within infected phagocytes of fixed CMV:GFP-Lc3 control and cyba knockdown embryos at 1, 2, and 8 hpi with approximately 1500 CFU. Data are shown as mean ± standard deviation (SD) obtained from three independent experiments (5–6 larvae per experiment per group). For 1 hpi, 205 infected phagocytes in 18 larvae analyzed. For 2 hpi, 188 infected phagocytes in 18 larvae were analyzed. For 8 hpi, 161 infected phagocytes in 17 larvae were analyzed. Two-way ANOVA with Bonferroni’s posttest was used. **** P < 0.0001, ** P < 0.01, ns – not significant. (C) Confocal photomicrographs are shown as maximum intensity projections of the Lc3-mediated response at 1 hpi in control (top panel) and cyba knockdown live lyz:RFP-GFP-Lc3 embryos infected with mCherry-labeled S. aureus. Scale bars: 10 µm. (D) Quantification of Lc3 associations with intracellular S. aureus within infected neutrophils of live lyz:RFP-GFP-Lc3 embryos at 1 hpi with approximately 1500 CFU. Data are shown as mean ± standard deviation (SD) obtained from three independent experiments (6 larvae per experiment per group). 96 infected neutrophils in 18 control larvae were analyzed. 92 infected neutrophils in 18 cyba knockdown larvae were analyzed. Unpaired two-tailed t-test was used. **** P < 0.0001)