JCS02529 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 - Fig. S1. Frames from example time-lapse experiments of adherent HeLa cells and non-adherent Jurkat cells undergoing apoptosis in response to UV treatment.Phase contrast (top) and annexin V fluorescence (bottom) images are shown from corresponding time-points. Bars, 10 mm.

• Supplemental Figure 2 - Fig. S2. Confocal maximum projections of apoptotic HeLa cells (anisomycin-treated) induced in the presence of various cytoskeletal poisons (images correspond to the data presented in Fig. 6). Cells are labelled with anti-KDEL (1D3) or anti-calnexin antibodies for the ER (red), with phalloidin or anti-tubulin for actin and microtubules respectively (green), and with DAPI (blue). Bar, 10 mm.

• Movie 1 - Movie 1. Example of early apoptotic blebbing (zeoisis). Time-lapse video of HeLa cells undergoing apoptosis in response to anisomycin (5 mg ml^–1) treatment. Cells were monitored by phase contrast time-lapse microscopy. Images were obtained at 60 second intervals. The video is played at 10 frames per second.

• Movie 2 - Movie 2. Biphasic blebbing in adherent cells. SW13.Cl-2 cells were induced into apoptosis by UV treatment, and were monitored by phase contrast time-lapse microscopy. The apoptotic cell is undergoing zeoisis at the onset then following a brief pause it initiates a second phase of blebbing. These late blebs are morphologically distinct. Images were obtained at 30 second intervals. The video plays at 10 frames per second.

• Movie 3 - Movie 3. Monophasic blebbing in non-adherent cells. Jurkat cells were induced into apoptosis by UV treatment, and were monitored by phase contrast time-lapse microscopy. The cell displays modest surface alterations during the early part of the video, then following a short static period, it begins a phase of dramatic blebbing. The blebs produced during this single phase are morphologically similar to late blebs in adherent lines. Images were obtained at 30 second intervals. The video plays at 10 frames per second.

• Movie 4 - Movie 4. Chromatin dynamics during apoptosis. HeLa cells were transiently transfected with the chromatin marker, HMGB1-YFP, then induced into apoptosis by UV treatment. Chromatin translocation into surface blebs occurs exclusively during the second, late phase of blebbing. The video is presented as an overlay of fluorescence (red) and phase contrast. Images were obtained at 120 second intervals. The video plays at 10 frames per second.

• Movie 5 - Movie 5. Effect of the ROCK inhibitor Y27632 on apoptotic blebbing. Y27632 inhibits zeiosis but is unable to completely prevent formation of late blebs. SW13.Cl-2 cells were induced into apoptosis by UV-treatment and were incubated with 100 mm Y27632. Cells were then monitored by phase contrast time-lapse microscopy. Images were obtained at 30 second intervals. The video plays at 10 frames per second.