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. 2026 May 27;6(5):1220–1238. doi: 10.1158/2767-9764.CRC-25-0648

Figure 5.

Figure 5.

AOH1996 disrupts centrosome organization and spindle integrity. A, Representative immunofluorescence microscopy images of HFK, HeLa, and CaSki cells treated with DMSO or 1 μmol/L AOH1996 for 24 hours, stained for β-tubulin (red), γ-tubulin (green), and DNA (DAPI, blue). In DMSO-treated cells, mitotic spindles and centrosomes appear organized and bipolar. In contrast, AOH1996-treated cells exhibit multipolar, shortened, and disorganized spindles with supernumerary centrosomes. B, Quantification of spindle length shows a significant reduction in AOH1996-treated cells across all lines, confirming spindle collapse or failure. C, Quantification of centrosome number per cell reveals a significant increase following AOH1996 treatment, most pronounced in cervical cancer (CaCx) cells. D, Chemical structures of AOH1996 and its structurally related analog AOH1996-8Nq, which contains a nitrogen substitution in the aromatic ring and lacks cytotoxic or mitosis-disrupting activity. E, Protein thermal shift assay of recombinantly expressed 6xHis PCNA with AOH1996-8Nq. A PCNA-stabilizing shift of 0.5°C at each concentration indicates binding interaction. F, Brightfield and immunofluorescence microscopy images of HeLa cells treated with 200 nmol/L AOH1996 or AOH1996-8Nq. AOH1996 causes cell rounding and centrosome/spindle disruption, whereas AOH1996-8Nq does not. G, Immunofluorescence microscopy of β-tubulin (red), γ-tubulin (green), and DAPI (blue) in HeLa cells. AOH1996 significantly decreases colocalization between γ-tubulin and β-tubulin, as shown by quantification (right). AOH1996-8Nq has no effect. Bottom, pixel-based colocalization maps show overlapping γ/β-tubulin signal (white). H, Western blot analysis of centrosome components (pericentrin and γ-tubulin), spindle protein (β-tubulin), and mitotic marker (p-H3) from HeLa cells treated with DMSO, AOH1996, or AOH1996-8Nq. AOH1996 decreases centrosomal protein levels and increases p-H3 abundance. AOH1996-8Nq does not alter these levels. Nucleolin serves as a loading control. Bar graph represents corresponding quantification. Bar graphs represent the mean ± SEM from three independent experiments. Statistical comparisons were performed using two-way ANOVA. ****, P < 0.0001; ***, P < 0.001; **, P < 0.0001.