Analysis of the apoptosis pathways induced by FcαRI targeting. (A) WT FcαRI and FcαRI_R209L/γ RBL transfectants were treated with 20 μM zVAD-fmk or solvent for 40 minutes before adding 10 μg/mL anti-FcαRI (A77) Fab. After 24 hours cells were analyzed for annexin V/PI double-positivity as described in “Materials and methods.” Values indicate the percentage of double-positive cells and are means ± SD of at least 3 separate experiments performed in duplicate. *P < .01. (B) Analysis of the mitochondrial pathway of apoptosis. FcαRI_R209L/γ RBL transfectants were incubated with the mitochondrial apoptosis pathway-inducing compound CCCP (200 μM) or with 10 μg/mL anti-FcαRI (A77) Fab or control (320) Fab. After 24 hours cells were stained with the mitochondrial membrane potential–sensitive dye DiOC₆ and analyzed by flow cytometry as described in “Materials and methods.” Induction of the mitochondrial pathway resulted in a strong loss of DiOC₆ staining. (C) Analysis of the “cleaved” (activated) form of caspase-3. FcαRI_R209L/γ cells were treated with 10 μg/mL A77 or control (320) Fab for the times indicated. Cells were then lysed, separated by SDS-PAGE, and transferred to PVDF membranes before immunoblotting with an antibody recognizing the cleaved form of caspase-3. To control for equal loading, blots were stripped and reprobed with antiscramblase (PLSCR) antibody plus goat anti–rabbit Ig-HRP. One representative experiment of 3 is shown. (D) Effect of SHP-1 siRNA treatment on FcαRI-mediated apoptosis in FcαRI_R209L/γ RBL transfectants. Analysis of relative SHP-1 expression levels by anti–SHP-1 immunoblotting in lysates from cells transfected with scrambled or SHP-1–specific siRNA (left panel). Analysis of the percentage of FcαRI-mediated apoptosis in FcαRI_R209L/γ cells transfected with scrambled siRNA or SHP-1–targeting specific siRNA (right panel). FcαRI_R209L/γ cells were transfected twice, 24 hours apart, with scrambled siRNA or SHP-1–targeting specific siRNA. After the second transfection, cells were treated with 10 μg/mL A77 or irrelevant control (320) Fab (right panel). Cells were then analyzed for hypoploid nuclei as described in “Materials and methods.” Numbers are the percentage of cells with hypoploid nuclei at 18 hours in a representative experiment.