HCT116 and HCT8 cells were treated with the indicated concentrations (0, 10, 20, and 40) of DHPITO for 12 h before being collected and lysed with radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 1% Nonidet P-40 (NP-40), 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 25 mM Tris-HCl (pH 7.4) and 1 mM EDTA (pH 8.0)) supplemented with protease and phosphatase inhibitors (cat no. 5892791001; Roche Diagnostics, Mannheim, Germany) on ice. The lysates were centrifuged at 12,000× g for 20 min, and the pellets and supernatants were collected to detect the assembled (cytoskeletal) and unassembled (cytosolic) forms of tubulin. Subsequently, the protein concentration of the supernatants was measured using a bicinchoninic acid (BCA) protein assay kit (cat no. P0010; Beyotime Institute of Biotechnology). A total of 50 μL 1× loading buffer was added to the pellets, which were then boiled for 10 min. An equal amount of each sample (30 μg) was loaded onto 10% and 15% SDS-polyacrylamide gel electrophoresis (PAGE) gels, followed by transfer to polyvinylidene difluoride (PVDF) membranes (MilliporeSigma). The membranes were blocked with nonfat dry milk (cat no. 9999S; Cell Signaling Technology, Inc., Danvers, MA, USA) for 1 h at room temperature; afterwards, they were incubated with the respective primary antibodies as referenced in the Reagents and Antibodies section above at 4 °C overnight, and subsequently incubated with IRDye® 800CW goat antimouse IgG (H + L) or IRDye® 680LT donkey antirabbit IgG (H + L) (LI-COR Biosciences, Lincoln, NE, USA) secondary antibody at room temperature in a dark environment for 1 h. Lastly, immunoreactivity was visualised using an Odyssey Two-Color Infrared fluorescence Imaging System (LI-COR Biosciences).
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