Figure 3.

AOH1996 induces mitotic arrest in cervical cancer (CaCx) cells. A, Cell-cycle analysis of HFKs and CaCx cell lines (HeLa, HeLa POLη, CaSki, and C33a) treated with 1 μmol/L AOH1996 for 24 or 48 hours. DNA content was measured by PI staining and flow cytometry. AOH1996 induces a G2/M arrest at 24 hours in all cell types. At 48 hours, CaCx cells accumulate in sub-G1 and >G2 populations, indicative of apoptosis and rereplication. B, Immunoblot of p-H3, a marker of mitotic chromatin condensation, from lysates of cells treated with 1 μmol/L AOH1996 for 24 hours. AOH1996 significantly increases p-H3 levels in CaCx cell lines but only modestly in HFKs, indicating selective induction of mitotic arrest in transformed cells. GAPDH is used as a loading control. Bar graph shows fold change relative to DMSO-treated controls. C, Mitotic spread assay of HFK, HeLa, and HeLa POLη cells treated with DMSO, 1 μmol/L AOH1996, or colcemid for 24 hours. Representative brightfield images show chromatin condensation characteristic of mitosis. Insets highlight mitotic figures. Quantification (right) shows the percentage of cells in mitosis. AOH1996 induces mitotic arrest in CaCx cells but not HFKs, whereas colcemid arrests both. D, Confocal immunofluorescence microscopy of HeLa and CaSki cells treated with DMSO or 1 μmol/L AOH1996 for 24 hours. Cells were stained with DAPI (blue) and anti–p-H3 (red) to identify mitotic cells (white arrows). Quantification (right) shows that >60% of AOH1996-treated cells are p-H3–positive, confirming widespread mitotic arrest. Bar graphs represent the mean ± SEM from at least three independent experiments. Statistical analysis by one-way ANOVA. **, P < 0.01; ***, P < 0.001; **, P < 0.0001.