Fig. 7

BRAF inhibitor sensitizes the 65-SR cells to sotorasib withdrawal. A The 65-SR cells were cultured with or without 5 μM sotorasib, 100 nM encorafenib, or both. The cells were counted on the indicated days. B Effects of encorafenib on the levels of p-ERK and ERK in the 65-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 in the 65-SR with or without 5 μM sotorasib and/or 100 nM encorafenib 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 65-SR cells cultured with or without 5 μM sotorasib, 100 nM encorafenib, or both. The yellow arrows denote the locations of nucleoplasmic bridges (D) or micronuclei (E). F Results of annexin V/PI flow cytometric assay on the 65-SR cells cultured with or without 5 μM sotorasib, 100 nM encorafenib, or both. G Schematic of the drug treatment of the 65-SR xenografts models. Sotorasib (20 mg/kg) was orally administered one day before and after tumor injection until the mean tumor volume reached at least 100 mm³. Next, the mice were divided into sotorasib +/encorafenib −, sotorasib-/encorafenib −, and sotorasib −/encorafenib + groups (n = 4–6 mice per group) and treated with the indicated drugs for 14 days. The concentration of encorafenib used was 20 mg/kg. H–J Tumor sizes (H) and body weights (J) of the mice were measured every 2 days. I At the end of experiment, the tumors were removed and photographed. Data are presented in terms of the mean ± standard deviation values of three cell culture replicates (A) or 4–6 mice (H, J). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001