Fig. 4

Sotorasib withdrawal induces premature mitotic entry and aberrant mitosis. A The MIA-SR and H23-SR cells were cultured with or without 5 μM sotorasib for 11 days and pulsed with iFluor 594-labeled EdU (red) for 4 h. p-Histone H3 (green) was detected through immunofluorescence staining. B The MIA-SR and H23-SR cells were cultured with or without 5 μM sotorasib for the indicated days and then subjected to immunofluorescence staining with γ-H2AX (orange), p-histone H3 (green), and cleaved caspase-3 (red) antibodies. C Representative images of immunofluorescence staining of the MIA-SR cells in the mitotic phase. The MIR-SR cells were cultured with or without 5 μM sotorasib for the indicated days and then stained with γ-tubulin (red) and β-tubulin (green). The quantification of abnormal mitosis was defined as more than one pair of centrosomes (γ-tubulin +) and mitotic spindles (β-tubulin +) in one cell. D Time-lapse microscopy images of the MIA-SR cells cultured with or without 5 μM sotorasib for the indicated days. The yellow arrows denote the indicated cell. E, F Representative images of immunofluorescence staining and quantification of nucleoplasmic bridges (E) and micronuclei (F) in the MIA-SR and H23-SR cells cultured with or without 5 μM sotorasib. The yellow arrows denote the locations of nucleoplasmic bridges (E) or micronuclei (F). A minimum of 200 nuclei per condition were analyzed (A–C, E, F). *P < 0.05, **P < 0.01, and ***P < 0.001