2.11 Western blot assay

HCT116 cells were inoculated overnight in 6-well plates. The cells were then exposed to test compounds for 48 h. After that, the cells were lysed on ice with a lysis buffer (Beyotime, Shanghai, China). The concentration of protein was measured using a BCA kit (Thermo Scientific, MA, United States). 30–50 μg of protein were deposited onto 10% SDS-PAGE gels before being transferred to polyvinylidene fluoride (PVDF) membranes. An hour was spent blocking membranes with 5% non-fat milk in Tris-buffered saline Tween 20 (TBST). The blots were incubated overnight at 4°C with primary antibodies (1:1,000), including rabbit antibodies p53 and PTEN (1:1000, Cell Signaling Technology, MA, United States), and mouse antibodies β-actin (1:3000, Merck, MO, United States) as listed in Supplementary Table S2. After three 15 min washes with TBST solution, the membranes were incubated for 1 h with secondary horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibodies (1:3,000, Thermo Scientific, MA, United States). Blots were then visualized using the ChemiDoc XRS + Imaging System (Bio-Rad, CA, United States) after the ECL reagent detection (GE Healthcare Life Sciences, Sweden). The bands were measured using Image J (NIH, United States). Each protein sample’s band intensities were normalized to its own internal standard proteins (β-actin). The quantitative results were presented as a multiple of the untreated control.