Methyl thiazolyl tetrazolium (mtt)

Cells were seeded at a density of 3 × 10³ cells/well in 96-well plates. After overnight stabilization, the cells were treated with either EPP (0.5–5 μg/mL) or its constituents, including PA, PB (ChemFaces), and PX (ChemFaces), for 72 h. Subsequently, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Duchefa, Haarlem, The Netherlands) solution (4 mg/mL) was added to the culture medium at a final concentration of 0.4 mg/mL and incubated for an additional 2 h. The culture medium was then carefully discarded and the insoluble MTT formazan was solubilized by adding 100 μL of DMSO. Cell viability was evaluated by measuring the absorbance of each well at 540 nm using a microplate reader (SpectraMax M3; Molecular Devices, San Jose, CA, USA).

The Griess reaction was performed to measure the concentration of nitrite in the medium as an indicator of NO production. RAW264.7 macrophage cells were cultured in a 96-well plate after seeding at a density of 4 × 10⁵ cells/well for 24 h in DMEM supplemented with 10% FBS, and stimulated with or without LPS (1 μg/mL, Sigma Aldrich, St. Louis, MO, USA) in the presence or absence of the compounds. After 24 h of incubation at 37 °C, 5% CO₂, the cell supernatant was reacted with equal volumes of Griess reagent solutions to determine nitrite production. Absorbance was measured with a microplate reader (Molecular Devices, Sanjose, CA, USA) at 540 nm. Cell viability was confirmed via MTT (Duchefa Biochemie, Haarlem, The Netherlands) assay. The supernatant was removed and medium containing MTT solution (5 mg/mL in phosphate-buffered saline) was added to each well and incubated for 2 h. The medium was removed, and 100 μL of dimethyl sulfoxide (Duchefa Biochemie, Haarlem, The Netherlands) was added to each well to dissolve the purple formazan product to obtain a colored solution. Absorbance was measured at 540 nm with a microplate reader.

Cells were seeded in 96-well culture plates at a density of 1 × 10⁴ cells/well (for HUVEC) or 4 × 10³ cells/well (for H1299). Following overnight stabilization, the cells were exposed to varying concentrations of HAT, EAT, and BAT (10–200 μg/mL) for 24 h. PC9 cells were seeded in 96-well culture plates at a density of 3 × 10³ cells/well and treated with erlotinib (0.1 μM or 1 μM) with or without HGF (20 ng/mL) for 72 h. Subsequently, a solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Duchefa, Haarlem, The Netherlands) was added to each well at 0.4 mg/mL. After 2 h of incubation at 37 °C, the culture medium was removed, and 100 μL of DMSO was added to dissolve the MTT formazan crystals. The cell viability was quantified by measuring the absorbance of the formazan solution at 540 nm using a microplate reader (SpectraMax M3; Molecular Devices, San Jose, CA, USA).