2.2. In vitro Cell Proliferation Assay

The anti-proliferation effect of neratinib on cells was assessed using the CellTiter-Blue (#G8081, Promega, Madison, WI, USA) cell viability assay or sulforhodamine B staining assay. In brief, 1 to 6 × 10³ cells/well were seeded in 96-well plates and treated the next day with neratinib with or without target kinase inhibitors for 5 days. The CellTiter-Blue reagent was added to the plates, and the optical density at 595 nm was determined using a VICTOR X3 plate reader (PerkinElmer, Waltham, MA, USA). After completing the CellTiter-Blue assay, the cells were fixed with 5% trichloroacetic acid at room temperature for 2 h and then stained with a 0.025% sulforhodamine B solution (#AAA1476914, Fisher Scientific, Waltham, MA, USA) for 30 min at room temperature. After washing with 1% acetic acid solution to remove the excessive sulforhodamine B solution, the stained cells were dissolved in 10 mM Tris buffer. Optical density was determined fluorometrically at excitation and emission wavelengths of 488 and 585 nm, respectively, using the VICTOR X3 plate reader. Growth inhibition graphs were generated, and IG₅₀s were calculated using nonlinear regression to fit the data to the log (inhibitor) vs. response (variable slope) curve using Prism software (V9.0, San Diego, CA, GraphPad, USA). The CalcuSyn program (V2, Biosoft, Cambridge, United Kingdom) was used to evaluate the synergistic anti-proliferation effect of neratinib in combination with kinase inhibitors.