Calcein am

Cell migration or invasion was determined using the Boyden chamber assay. Cell migration was performed using uncoated transwell chambers, while cell invasion was performed using transwell chambers coated with matrigel matrix (Corning, Fisher Scientific, Waltham, MA). Briefly, 200 μl of cells were added at 5 × 10⁵ cells/ml to the upper compartment of a transwell chamber with a 0.8 μm pore size (Corning). Complete DMEM media was added to the well which the chamber is inserted into. After incubation at 37°C for 24 h (for Panc-48) or 48 h (for MiaPaCa-2), cells were labeled with calcein-AM (Molecular Probes, Eugene, OR) and fixed with 4% paraformaldehyde in PBS. The total amount of cells in the upper chamber as well as cells that had migrated through the membrane was first quantified by reading the fluorescence of calcein AM-labeled cells at an excitation wavelength of 485 nm and an emission wavelength of 520 nm using a microplate reader (Bio-Tek, Winooski, VT), and then cells in the upper chamber were removed via scraping with a cotton tip and washing with PBS, and the amount of cells left on the bottom surface of the membrane, the cells that had migrated, were quantified again by reading fluorescence at the same wavelengths. Migration rate was presented as: fluorescence reading before scraping (total cells)/fluorescence reading after scraping (migrated cells) x 100%.

Viability and necrosis were performed as reported in earlier studies^{28 (link),86 (link),87 (link)}. Briefly, NHBE or A549 were seeded in 96-well cell culture plates and incubated for up to 6 h (10.000 cells/well), 24 h (10.000 cells/well), and 7 days (2.500 cells/well) with GO, FLG, or sFLG at increasing concentrations (0.05, 0.5, 5, 50, and 100 µg/mL). For FBS starvation tests, A549 cells were pre-cultured for 6 h or 24 h in a serum-free medium. Cells were then incubated with 10 μg/mL ethidium bromide (EtBr) (#46,067; Sigma-Aldrich) and 1 μM Calcein-AM (#C34852; Thermo Fisher). Viable cells, stained with green Calcein-AM, and necrotic cells, stained with red EtBr, were determined by fluorescence microscopy using a Cytation 5 Cell Imaging Multi-Mode Reader (20 × objective; BioTek) and analyzed with ImageJ 1.53. After image acquisition in living cells, samples were fixed and permeabilized in cold methanol for 4 min and then stained with 1 μg/mL Hoescht (#861,405; Sigma-Aldrich) to visualize DNA. Apoptosis was quantified by qualitative methods, as reported in earlier studies^{28 (link)}. Results are presented as the number of cells per field or as a percentage of necrotic or apoptotic cells vs. total (n = 3).