Fig. 6

Low-dose BRAF inhibitor aggravates sotorasib withdrawal–induced DNA damage and cell death. A The MIA-SR and H23-SR cells were cultured with or without 5 μM sotorasib, 100 nM encorafenib, or both. The cells were counted on the indicated days. Data are presented in terms of mean ± standard deviation values of three cell culture replicates. B Effects of encorafenib treatment on the level of p-ERK, ERK, p-RPA32, p-CHEK1, and p21 in the MIA-SR cells cultured with or without 5 μM sotorasib; protein levels were measured through Western blotting. C Representative images of immunofluorescence staining and quantification of γ-H2AX staining pattern performed using the MIA-SR and H23-SR cells cultured with or without 5 μM sotorasib, 100 nM encorafenib, or both for the indicated days. −: < 5 foci/cell, +: > 5 foci/cell, and + + : pan-nuclear expression. D, E Representative images of immunofluorescence staining and quantification of nucleoplasmic bridges (D) and micronuclei (E) in the MIA-SR cells cultured with or without 5 μM sotorasib, 100 nM encorafenib, or both. The yellow arrows indicate the locations of micronuclei (D) or nucleoplasmic bridges (E). F Results of annexin V/PI flow cytometric assay performing using the MIA-SR cells cultured with or without 5 μM sotorasib, 100 nM encorafenib, or both. A minimum of 200 nuclei per condition were analyzed (C–E). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001