The anticancer effects of Arf1-targeting compounds. (A) The effects of Arf1-targeting compounds (9b, 9c, 10a, 10b, 10c, and 10d in a range dose from 0 to 20 µM) on HN12 cell viability were determined by CellTiter-Glo® Luminescent cell viability assay after 72 h treatment. (B) The dose-dependent cytotoxicity of compound 10b in three HNSCC cell lines (HN4, HN12, and HN31) was determined by CellTiter-Glo® Luminescent cell viability assay after 72 h treatment. (C,D) The effects of 20 µM of compound 10b on HN12 cell viability in SeedEZ was determined after 72 h treatment. Representative images of DAPI staining and quantitative data of cell viability measured by alamarBlue are shown in (C,D), respectively. (E,F) Arf1 protein levels and its activity in HN12 and HN4 cells treated with or without compound 10b were determined by GGA3 pull-down assay and Western blotting. A representative result and quantitative data from three independent experiments are shown in (E,F), respectively. * p < 0.05; ** p < 0.01.