(A) C5 induces a drop in ▵ψm of HL60 cells.
HL60 (1 × 106 cells/mL) were exposed to 150 μg/mL C5 for 8 hours and loaded with membrane-sensitive probe (40 nmol/L) at 37°C for 30 minutes, washed, and analyzed by flow cytometry, as described in “Materials and methods.” (B) C5 triggered cytosolic translocation of mitochondrial Cyt C in HL60 cells. HL60 (30 × 106 cells) were treated with 50-150 μg/mL C5 for 18 hours, and cytosolic fractions were subjected to SDS-PAGE electrophoresis, transferred to polyvinylidene difluoride membrane, and subjected to Western blot analysis for Cyt C, as described in “Materials and methods.” (C) M14 cells (1 × 103) were grown on coverslips and treated with 150 μg/mL C5 for 12 hours, and Cyt C localization was determined by confocal microscopy using anti-Cyt C, as described in “Materials and methods.”
