Cell Viability Assay Protocol

Cells were seeded into microtiter plates (Life Sciences, Corning, NY, USA) at a concentration of 5 × 10³ cells/100 µL/well in full medium and allowed to attach overnight, followed by the incubation in fresh full medium with or without the test substances (at concentrations of 0.005, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, and 1 µg/mL for nintedanib and 1, 10, 50, 125, 250, 500, 750, and 1000 µg/mL for the other 3 substances) for 48 hours. The stock solution of MTT (5 mg/mL in PBS; Gibco by Lifetechnologies, Carlsbad, CA, USA) was added at a dilution of 1:5 (v/v) and the cells were incubated further for 3.5 hours.⁶⁴ (link)^,65 (link) Measurement of absorbance (UV/visual absorption spectroscopy in optical density [OD]) was performed using a spectrophotometer (Molecular Devices GmbH, Biberach an der Riss, Germany) at 570 nm⁶⁴ after dissolving the MTT crystals with DMSO.⁶⁶ (link) Negative controls were performed by measuring wells without cells but with the addition of MTT solution to prevent false positive results.