Figure 5
Preferential apoptosis execution of HeLa.S-Fucci cells at G1 phase after treatment with the agonistic anti-Fas antibody. Cells were arrested at the border of G1 and S phase with a double-thymidine block, followed by release to enter S phase in the absence or presence of 0.5 μg/ml of the agonistic anti-Fas antibody or 50 μM etoposide (VP-16), and were observed with the time-lapse fluorescence microscope at 15-min intervals (see Supplementary Movies S4–S6). (a) Mitotic cells showing smooth round morphology with mAG1 were counted on the movies and were plotted over time. The plots showed the average number of mitotic cells from at least six fields. (b) Total cell number including normal, apoptotic, and mitotic cells were counted on the movies and the images constructing the movies. The plots showed the average number of cells from at least three fields. (c) Cell viability after treatment with the agonistic anti-Fas antibody was assessed morphologically on the movies and the images constructing the movies by counting flat and well-attached cells as viable cells, and shrunk or fragmented cells as dead cells. At least 120 cells were counted for each measurement in one movie. The data (mean±S.D.) were obtained from at least six movies. (d) The average number of apoptotic cells per one area of 0.58 mm2 at each time point was obtained from the data in panel c. (e) A histogram showed the average number of apoptotic cells per one field of 0.58 mm2. The data were obtained from panel d. (f) Cell viability after treatment with etoposide (VP-16) was assessed morphologically as described in panel c. (g) The average number of apoptotic cells per one field of 0.58 mm2 at each time point was obtained from the data in panel f. (h) A histogram showed the average number of apoptotic cells per one field of 0.58 mm2. The data were obtained from panel g
