Cells were seeded onto glass slides. After 24 hours, the cells were fixed with 4% paraformaldehyde for 10 min at room temperature, permeabilized with 0.1% Triton X-100, and incubated with 5% bovine serum albumin. The cells were stained with SMAD4 (1:50, D3R4N; Cell Signaling Technology) for 2 hours at room temperature followed by incubation with Alexa Fluor 488–conjugated secondary antibody (1:1000, Invitrogen) or with Ki67 Alexa Fluor 488–conjugated antibody (1:100, D3B5; Cell Signaling Technology) for 2 hours at room temperature. Nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories). Staining was analyzed using an Olympus IX81 fluorescence microscope and the cellSens Dimension software.

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