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. 2021 May 27;22(11):5706. doi: 10.3390/ijms22115706

Figure 1.

Figure 1

Combination of OSI-906 and ZM447439 generates enlarged cells with aberrant-shaped nuclei. HeLa S3 cells were treated with 3 µM OSI-906, 1 µM ZM447439, or their combination for 3 days. The cells were fixed with methanol for 5 min at −30 °C (a,c,d) or 4% formaldehyde for 20 min at room temperature (b). (a) The fixed cells (Day3) were stained for cleaved caspase-3 (red) and DNA (cyan). Representative images of cells treated with OSI-906, ZM447439, or their combination (OSI + ZM) are shown. Dimethyl sulfoxide (DMSO) was used as solvent control. Yellow arrows indicate cleaved caspase-3–positive cells. Scale bar, 20 µm. (b) On day 1 and day 2, the cells were fixed and stained for cleaved caspase-3 and DNA. The number of cleaved caspase-3–positive cells were counted and plotted as the mean ± SD calculated from three independent experiments (n > 201). p-values were calculated using Tukey’s multiple comparison test (Day1) and the Games–Howell multiple comparison test (Day2). * p < 0.05; *** p < 0.001. (c) The fixed cells were stained for DNA (cyan). Representative images of cells treated with the combination (OSI + ZM) for 1–3 days are shown. Scale bar, 20 µm. (d) The fixed cells (Day3) were stained for γ-tubulin (green) and DNA (red). Representative images of γ-tubulin in interphase (left) and M phase cells (right) treated with the combination (OSI + ZM) are shown. Scale bar, 10 µm.