Gen5 software 3

A549 cells were seeded in clear-bottom black 96-well plates (Corning, New York, NY, USA) (10⁴ cells/well). After overnight incubation, cells were treated with either a vehicle control (0.5% DMSO in culture medium), TNF-α only (20 ng/mL), or 1.5 µM patulin + TNF-α (20 ng/mL) for 20 min. Cells were then rinsed with DPBS (Thermo Fisher scientific), fixed with 4% paraformaldehyde for 10 min, and permeabilized 5 min with a 0.1% Triton X-100 DPBS solution (DPBST). Blocking was then performed for 30 min with 1% BSA in DPBST, and cells were incubated overnight at 4 °C with the rabbit anti-p65 antibody (Cell Signaling Technology, Danvers, MA, USA). After rinsing three times with DPBS, cells were incubated for 1 h at room temperature in the dark with an anti-rabbit antibody (Cell Signaling Technology), and counterstained with 0.1 µg/mL DAPI for 1 min. After rinsing three times in DPBS, fluorescent pictures were taken on the Cytation 3 imaging multimode reader (Biotek). NF-κB p65 nuclear translocation was quantified by measuring the fluorescence intensity of the secondary antibody in the nuclear zones defined by DAPI staining, using the Gen5 software 3.0 (Biotek).

The scratch assay was used to determine cell migration as previously described [27 (link)]. Briefly, A549 cells were seeded in a 96-well plate (1.5 × 10⁴ cells/well) and grown until confluence. A scratch was then drawn in each well using a 200 µL pipette tip and debris were removed by rinsing cells with DPBS. Cells were then treated with increasing concentrations of patulin or with DMSO alone (0.5% max). Pictures were taken quickly after treatment and after 24 h of incubation at 37 °C in a 5% CO₂ atmosphere. Widths of the wounds were measured at both time points using the Gen5 software 3.0 (Biotek) and percentage of space recovery after 24 h was obtained using the formula: % recovery = 100 − (width 24 h/width 0 h × 100). Inhibition of cell migration was quantified by comparing the percentage of space recovery between the cells treated with patulin and the cells treated with DMSO alone.