Cell proliferation assays were carried out using Cell Counting Kit 8 (CCK8) (Dojindo). HCT116 cells were plated in 96-well plates at a density of 2 × 103 cells per well and cultured in serum-free medium. Twenty-four hours later, the cells were treated with the indicated concentration of N8-acetylspermidine. At the indicated time points, cell numbers were measured using CCK8 according to the manufacturer’s instructions. For cell proliferation immunostaining, HCT116 cells were fixed with 4% paraformaldehyde in PBS, permeabilized by 0.1% Triton X-100, treated with 2%BSA in PBS for 10 min at room temperature, respectively. Then, cells were incubated with Ki-67 antibody (eBioscience, 14–5698-82) and fluorescence-conjugated secondary antibody (Thermo Fisher Scientific, A-11006), and observed under fluorescence microscope.

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