Figure 3
Figure 3. Assembly and activation of the NADPH oxidase in response to serum-opsonized S aureus is partially dependent on binding of p40phox to PtdIns3P, but fully dependent on Rac. (A) Primed neutrophils (1 × 106) isolated from WT (black squares/bars) or p40phoxR58A/R58A (R58A, dark gray diamonds/bars) genotypes (i,iii); or from WT (black squares/bars), Rac1 knockdown on a WT background (Rac1KD, gray diamonds/bars), Rac2−/− (Rac2KO, light gray triangle/bar), and Rac1 knockdown on a Rac2 KO background (Rac1KD/Rac2KO, open circles/bar; ii-iii) were preincubated with luminol/HRP, before addition to serum-S aureus as described in “Measurement of ROS production.” Total ROS responses were measured by chemiluminesence and recorded on a 96-well plate using a Berthold Microlumat Plus luminometer as described in “Measurement of ROS production.” All incubations were performed in at least duplicate. Shown are data (mean ± range) from 1 experiment representative of at least 3, expressed as RLU/s (i-ii), as well as accumulated light emission (ROS response) over 20 minutes for a combination of ≥ 3 experiments (mean ± SEM), expressed as a percentage of the response in WT mouse neutrophils (iii). *P < .0001, paired Student's t test (WT/R58A), 1-way analysis of variance (ANOVA) (WT, Rac1KD, Rac2KO, Rac1KD/Rac2KO), compared with WT response. (B) Primed BMNs from the indicated genetic backgrounds, after phagocytosis of serum-S aureus were fixed and stained and imaged for p67phox as described in Figure 2A. Phagosomal p67phox fluorescence was quantified as described in panel A and is expressed (mean ± SEM) as a percentage of WT levels, *P < .0001, 1-way ANOVA. Arrows indicate the position of S aureus bacteria in the DIC images. (C) For quantification of cytosolic (1) and phagosomal (2) fluorescence, mouse BMNs expressing p67phox WT-GFP on a WT or a p40phoxR58A/R58A genetic background or expressing p67phox H69E-GFP on a WT genetic background were primed and incubated with S aureus as described in Figure 2B. Images were recorded on an Olympus CellR epifluorescence microscope equipped with an Olympus 1 × 2-UCB camera and using a 60× oil objective, as further described in “Live imaging.” Images were recorded for 15 minutes in the GFP channel and DIC channel (not shown) at 4-second intervals and at an exposure time of 750 milliseconds. Representative images are shown. Fluorescence around the phagosomes was quantified using ImageJ software, as described in “Live imaging” and supplemental Figure 9. Data are presented as a ratio of MFI of phagosome:cytosol. The gray transparent area represents a ratio of phagosome:cytosol of 1.0 or lower, representing no phagosomal translocation. Data are mean ± SEM (n ≥ 19 phagosomes in at least 5 different neutrophils per construct/genetic background). *P < .0001 as determined by Student t test between WT and R58A, and between WT and H69E.

Assembly and activation of the NADPH oxidase in response to serum-opsonized S aureus is partially dependent on binding of p40phox to PtdIns3P, but fully dependent on Rac. (A) Primed neutrophils (1 × 106) isolated from WT (black squares/bars) or p40phoxR58A/R58A (R58A, dark gray diamonds/bars) genotypes (i,iii); or from WT (black squares/bars), Rac1 knockdown on a WT background (Rac1KD, gray diamonds/bars), Rac2−/− (Rac2KO, light gray triangle/bar), and Rac1 knockdown on a Rac2 KO background (Rac1KD/Rac2KO, open circles/bar; ii-iii) were preincubated with luminol/HRP, before addition to serum-S aureus as described in “Measurement of ROS production.” Total ROS responses were measured by chemiluminesence and recorded on a 96-well plate using a Berthold Microlumat Plus luminometer as described in “Measurement of ROS production.” All incubations were performed in at least duplicate. Shown are data (mean ± range) from 1 experiment representative of at least 3, expressed as RLU/s (i-ii), as well as accumulated light emission (ROS response) over 20 minutes for a combination of ≥ 3 experiments (mean ± SEM), expressed as a percentage of the response in WT mouse neutrophils (iii). *P < .0001, paired Student's t test (WT/R58A), 1-way analysis of variance (ANOVA) (WT, Rac1KD, Rac2KO, Rac1KD/Rac2KO), compared with WT response. (B) Primed BMNs from the indicated genetic backgrounds, after phagocytosis of serum-S aureus were fixed and stained and imaged for p67phox as described in Figure 2A. Phagosomal p67phox fluorescence was quantified as described in panel A and is expressed (mean ± SEM) as a percentage of WT levels, *P < .0001, 1-way ANOVA. Arrows indicate the position of S aureus bacteria in the DIC images. (C) For quantification of cytosolic (1) and phagosomal (2) fluorescence, mouse BMNs expressing p67phox WT-GFP on a WT or a p40phoxR58A/R58A genetic background or expressing p67phox H69E-GFP on a WT genetic background were primed and incubated with S aureus as described in Figure 2B. Images were recorded on an Olympus CellR epifluorescence microscope equipped with an Olympus 1 × 2-UCB camera and using a 60× oil objective, as further described in “Live imaging.” Images were recorded for 15 minutes in the GFP channel and DIC channel (not shown) at 4-second intervals and at an exposure time of 750 milliseconds. Representative images are shown. Fluorescence around the phagosomes was quantified using ImageJ software, as described in “Live imaging” and supplemental Figure 9. Data are presented as a ratio of MFI of phagosome:cytosol. The gray transparent area represents a ratio of phagosome:cytosol of 1.0 or lower, representing no phagosomal translocation. Data are mean ± SEM (n ≥ 19 phagosomes in at least 5 different neutrophils per construct/genetic background). *P < .0001 as determined by Student t test between WT and R58A, and between WT and H69E.

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