MTT assay

Cells were treated with PI3K inhibitor LY294002 (Selleck, S1105) or copanlisib (Selleck, S2802), Wnt pathway inhibitor ICG001 (Selleck, S2662) or mebendazole (Selleck, S4610), or a combination of two agents for the indicated period of time. LY294002 is an inhibitor of PI3Kα/δ/β, whereas copanlisib is an inhibitor of PI3Kα and PI3Kδ. ICG001 is an inhibitor of β-catenin, whereas mebendazole is an inhibitor of Traf2- and Nck-interacting kinase (TNIK), which interacts with TCF4 to regulate Wnt downstream targets, and 5-Aza-2′-deoxycytidine (5-aza) is the DNA methylation inhibitor. To evaluate the effect of PI3K inhibitors or Wnt pathway inhibitors, cells were seeded in 96-well plates at a density of 1500 cells per well. After an overnight incubation, cells were treated under the indicated conditions. To evaluate the effect of 5-aza, seeded cells were treated with 5-aza at a final concentration of 2.5 and 5 μM, respectively, with Aza-containing medium being changed every 24 h. At the end of the treatment, 20 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was added to each well, after which the plates were incubated at 37 °C for 4 h. Next, the supernatants were aspirated carefully, after which formazan crystals were dissolved in DMSO. Finally, the absorbance was measured at 490 nm. The concentration of each PI3K inhibitor, Wnt pathway inhibitor, and combination that produced 50% growth inhibition (IC50) was calculated using a relative survival curve 48 h after treatment. A drug combination experiment was performed by setting serial concentrations of one drug as above with a fixed concentration (IC25 or IC50) of another drug. Furthermore, the value of combination index (CI) calculated by the CalcuSyn software was used to determine the synergism between drug combinations [26–28].