Cell death and HA induced by GA101 is dependent on actin polymerization. (A) Time-lapse microscopy of Raji cells following treatment with GA101. Cells were suspended in phenol red-free media containing 7-aminoactinomycin D (7-AAD, 5 μg/mL) as a marker for cell death, and treated with GA101 (10 μg/mL) at time 0. Phase-contrast and fluorescence images were captured every 5 minutes and overlaid, with a sample of images captured at different time points shown (scale bar, 20 μm). Cellular adhesion was followed by cell swelling, loss of plasma membrane integrity, and cell death. Arrow marks a morphological control for apoptosis shown on the top left of the image. (B) Morphological pattern of cell death induced by GA101. Images show adhering cells showing gross cytoplasmic disintegration and loss of plasma membrane integrity after treatment with GA101. Staurosporine (STSP) was used as a morphological control for apoptosis. (C) Cells were incubated with inhibitors of actin polymerization (cytochalasin D and latrunculin B, 10μM) before treatment with mAb. Cell death and homotypic adhesion were analyzed 4 hours after treatment. Disruption of the actin cytoskeleton significantly inhibited cell death induced by GA101 and HA as shown in the example with latrunculin B (scale bar, 100 μm; *P < .02 **P < .01).