4.1. GLP-1 Secretion from NCI-H716 Cells

Briefly, NCI-H716 cells were seeded at 0.5 × 10⁶ cells/well in 24-well microplates coated with Matrigel and incubated for 48 h. The cells were then washed twice with phosphate-buffered saline (PBS) and incubated with PBS containing either GEF (0, 0.1, 0.3, 1, 3, 10, 30, and 100 µg/mL), LPA (5 µM), or glucose (200 mM) at 37 °C for 1 h. For the inhibition of GLP-1 release, we utilized Ki16425 (10 µM) and U73122 (5 µM) with or without GEF (100 µg/mL). The supernatants were collected and either immediately assayed or frozen to later perform analysis using a GLP-1 active ELISA kit (Millipore, Billerica, MA, USA) at an excitation/emission wavelength of 355/460 nm. The values of secreted GLP-1 were corrected by measuring the amount of protein using the BCA protein assay.