Enzyme linked immunosorbent assay plate reader

A total of 500 mg of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) powder (Sigma-Aldrich, USA) was dissolved in 100 ml of PBS to a final concentration of 5 mg/ml (0.5% MTT). The solution was stored in the dark at -20°C. A549 cells in the exponential growth phase were seeded in 96-well plates (Coring, USA) at 5×10³ cells (100 µl) per well and then incubated at 37°C in 5% CO₂ for 24 hours. Different concentrations of P3, Caerin 1.1, and Caerin 1.9 were added to the 96-well plates to reach final concentrations of 1, 5, 10, 15, 20, 25, 30, and 40 μg/ml. The experiment was done in triplicate and included control wells. The cells were cultured overnight. Once the drug’s effect was visible under the microscope, 10 µl of MTT solution was added to each well, and the cells were cultured for another 4 hours. Next, the supernatant was carefully aspirated, 150 µl of dimethyl sulfoxide solution (Sigma-Aldrich, USA) was added to each well, and the 96-well plates were placed on a shaker at low speed for 10 minutes. The absorbance at 570 nm was measured in an enzyme-linked immunosorbent assay plate reader (Thermo Scientific, USA). GraphPad Prism v9.3.0 was used calculate the half-maximal inhibitory concentration (IC₅₀) of each drug and the survival rate (%).

To optimize the safe working concentration of each inhibitor, its cytotoxicity was evaluated as described previously (Liu et al., 2019 (link)). GS cells (1 × 10⁴) were cultured in a 96-well plate (Corning, New York, United States) for 24 h at 28°C, and then incubated with different concentrations of each inhibitor (15 μM CPZ, 20 μM dynasore, 2 mM MβCD, 100 μM genistein, 40 μM EIPA, 20 μM IPA-3, 20 μM ML-7, 200 μM NSC23766, 1 μM rottlerin, 20 μM CQ, 400 mM NH₄Cl, or 6 μM cytoD) at 28°C for 4 h. Untreated GS cells were used as the control group. CCK-8 solution (20 μL; Beyotime, Shanghai, China) was added to the cells, and they were incubated at 28°C for 4 h. The absorbance at 450 nm was measured with an enzyme-linked immunosorbent assay plate reader (Thermo, Waltham, MA, United States), according to the manufacturer’s instructions. The results of three independent experiments are presented as means ± standard deviations (SD). The working concentration of each inhibitor used in this study caused no significant cytotoxicity.

We performed cell viability assays using a TACS XTT assay kit (R&D Systems). Briefly, cells were plated in 96-well plates (1×10⁴/well) and were treated with PDGF-AA or LY294002 for 24 hours. We added 50 μL of XTT working solution (combining XTT reagent with XTT activator) to each well. After incubating the cells for 6 hours, we obtained the absorbance values at 490 nm with a reference correction at 630 nm in an enzyme-linked immunosorbent assay plate reader (Thermo Scientific). Data were obtained from three independent assays in triplicate.
We performed colony formation assays as described previously [16 (link)]. Only colonies containing more than 50 cells were scored. Data shown are mean number of colonies observed in six randomly chosen microscopic fields (magnification ×200).