Figure 6
Defective dectin-1–mediated Rac1 activation and phagocytosis in XIAP-deficient human macrophages are corrected by resolvin D1. (A) Impaired Rac1 activity in XIAP-knockout human macrophages. Control (Ctrl) and XIAP-knockdown (small interfering XIAP) human macrophages, with or without active Rac1 (Rac1V12) transfected, were incubated with curdlan (100 μg/mL) and harvested at 0 and 10 minutes. The activation of Rac1 was measured by the G-LISA Rac1 activation assay kit. (B-C) Defective phagocytosis of C albicans in XIAP-knockdown human macrophages and restoration by Rac1V12 expression. Control and XIAP-knockdown human macrophages, with or without Rac1V12 transfected, were incubated with fluorescein isothiocyanate-conjugated heat killed C albicans (FITC-HKCA) for 30 minutes. Cells were collected at 0 and 30 minutes and analyzed by (B) confocal microscopy and (C) flow cytometry. (B) Cells were fixed, permeabilized, and stained with Alexa Fluor 594-Phalloidin (for F-actin) and 4′,6 diamidino-2-phenylindole for confocal microscopy. Bar indicates 20 μm. (C) Cells were also treated with trypan blue to quench the extracellular associated FITC, and the internalized FITC-conjugated bead was determined by flow cytometry. Gray shadow, fluorescence-activated cell sorting (FACS) profile at 0 minutes; blue and red curves, uptake of FITC-HKCA at 30 minutes; orange curve, internalization of FITC-HKCA at 30 minutes by Rac1V12-transfected macrophages. (D) Resolvin D1 restores Rac1 activity in XIAP-deficient human macrophages. Control and XIAP-knockdown human macrophages were incubated with curdlan (100 μg/mL) with or without AT-resolvin D1 (RvD1, 100 nM) pretreatment. Cells were harvested at 0 and 10 minutes, and the activation of Rac1 was measured. (E-F) Resolvin D1 restores phagocytosis of C albicans in XIAP-knockdown human macrophages. Control and XIAP-knockdown human macrophages, pretreated with vehicle or RvD1 (100 nM), were incubated with FITC-HKCA for 30 minutes. Cells were collected at 0 and 30 minutes and analyzed by (E) confocal microscopy and (F) flow cytometry. Gray shadowed curve, FACS profile at 0 minutes; blue and red curves, internalization of FITC-HKCA at 30 minutes; green curve, internalization of FITC-HKCA at 30 minutes by RvD1-treated macrophages. *P < .05 and **P < .01 for paired Student t test. Confocal images were obtained a Zeiss LSM 780 confocal microscope (Jena, Germany) with an objective lens of plan-Apochromat 63×/1.4 Oil DIC M27 at room temperature. 4′,6 Diamidino-2-phenylindole fluorescence was excited at 405 nm and collected at 420 to 470 nm. FITC fluorescence was excited at 488 nm and collected at 500 to 550 nm. Cy3 fluorescence (transfection indicator, not shown in figure) was excited at 561 nm and collected at 568 to 629 nm. Alexa Fluor 594 fluorescence was excited at 633 nm and collected at 641 to 758 nm. The pinhole was at 61.7 μm, with an 11.4-μm section. Images were taken by an AxioCam digital microscope camera (Zeiss) using Carl Zeiss software Zen 2010B SP1, version 6.0.

Defective dectin-1–mediated Rac1 activation and phagocytosis in XIAP-deficient human macrophages are corrected by resolvin D1. (A) Impaired Rac1 activity in XIAP-knockout human macrophages. Control (Ctrl) and XIAP-knockdown (small interfering XIAP) human macrophages, with or without active Rac1 (Rac1V12) transfected, were incubated with curdlan (100 μg/mL) and harvested at 0 and 10 minutes. The activation of Rac1 was measured by the G-LISA Rac1 activation assay kit. (B-C) Defective phagocytosis of C albicans in XIAP-knockdown human macrophages and restoration by Rac1V12 expression. Control and XIAP-knockdown human macrophages, with or without Rac1V12 transfected, were incubated with fluorescein isothiocyanate-conjugated heat killed C albicans (FITC-HKCA) for 30 minutes. Cells were collected at 0 and 30 minutes and analyzed by (B) confocal microscopy and (C) flow cytometry. (B) Cells were fixed, permeabilized, and stained with Alexa Fluor 594-Phalloidin (for F-actin) and 4′,6 diamidino-2-phenylindole for confocal microscopy. Bar indicates 20 μm. (C) Cells were also treated with trypan blue to quench the extracellular associated FITC, and the internalized FITC-conjugated bead was determined by flow cytometry. Gray shadow, fluorescence-activated cell sorting (FACS) profile at 0 minutes; blue and red curves, uptake of FITC-HKCA at 30 minutes; orange curve, internalization of FITC-HKCA at 30 minutes by Rac1V12-transfected macrophages. (D) Resolvin D1 restores Rac1 activity in XIAP-deficient human macrophages. Control and XIAP-knockdown human macrophages were incubated with curdlan (100 μg/mL) with or without AT-resolvin D1 (RvD1, 100 nM) pretreatment. Cells were harvested at 0 and 10 minutes, and the activation of Rac1 was measured. (E-F) Resolvin D1 restores phagocytosis of C albicans in XIAP-knockdown human macrophages. Control and XIAP-knockdown human macrophages, pretreated with vehicle or RvD1 (100 nM), were incubated with FITC-HKCA for 30 minutes. Cells were collected at 0 and 30 minutes and analyzed by (E) confocal microscopy and (F) flow cytometry. Gray shadowed curve, FACS profile at 0 minutes; blue and red curves, internalization of FITC-HKCA at 30 minutes; green curve, internalization of FITC-HKCA at 30 minutes by RvD1-treated macrophages. *P < .05 and **P < .01 for paired Student t test. Confocal images were obtained a Zeiss LSM 780 confocal microscope (Jena, Germany) with an objective lens of plan-Apochromat 63×/1.4 Oil DIC M27 at room temperature. 4′,6 Diamidino-2-phenylindole fluorescence was excited at 405 nm and collected at 420 to 470 nm. FITC fluorescence was excited at 488 nm and collected at 500 to 550 nm. Cy3 fluorescence (transfection indicator, not shown in figure) was excited at 561 nm and collected at 568 to 629 nm. Alexa Fluor 594 fluorescence was excited at 633 nm and collected at 641 to 758 nm. The pinhole was at 61.7 μm, with an 11.4-μm section. Images were taken by an AxioCam digital microscope camera (Zeiss) using Carl Zeiss software Zen 2010B SP1, version 6.0.

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