M1025

Exponentially growing cells were resuspended to 5 × 10⁴/mL. The cell suspension was sorted into 96-well plates at 100 μL per well till the cells reaching a density of 5 × 10³ cells/well (the marginal pores were filled with sterile phosphate-buffered saline (PBS) to eliminate the potential edge effect). Three duplicated wells were set for each group. The plates were incubated in a 37°C incubator with 5% CO₂. One plate was taken out every 24 hours for measurement. In brief, each well was filled with 20 μL MTT solution (M1025, Solarbio), and the cells were incubated for another 4 hours. Then, the culture medium in wells was discarded, and each well was further loaded with 150 μL dimethyl sulphoxide to fully dissolve the crystal violets. Then, the optical density at 570 nm of each well was determined using a microplate reader (Varioskan LUX, Thermo Fisher Scientific). The obtained data were analyzed to produce an MTT proliferation curve.

An MTT assay (M1025, Solarbio, Beijing, China) was performed to identify the IC₂₅ and IC₅₀ (quarter and half maximal inhibitory concentration, respectively) of each drug in each KRAS-mutant NSCLC cell line. Cells were seeded in 96-well plates at 5 × 10³ cells per well and allowed to grow overnight at 37°C in an atmosphere containing 5% CO₂. Thereafter, the cells were subjected to individual or combined drug treatment at the specified drug concentrations. After 48 hours of culture with the drug(s), 20 mL of MTT reagent (5 mg/mL) was added to each well and the plate was further incubated for 4 hours. The liquid was removed from the wells and 150 mL of dimethyl sulfoxide was added to each well. After 10 minutes of gentle shaking, the absorbance of the wells was measured using a plate reader (AMR-100, Allsheng, Hangzhou, China) at 490 nm.