Apoptosis Mechanisms Induced by Compound 3b in Lung Cancer Cells

To assess which death mechanisms our compounds induced, cell apoptosis kit Annexin V/Propidium iodide (PI) double staining uptake (Invitrogen, Life Technologies, Italy) was used. 3b compound was selected as lead and employed in our assay. Human lung cancer cells (SK-MES 1) were seeded at the density of 8 × 10⁵ cells/ml in 6-well plates (Corning, United States) with a complete medium (described in cell culture section). After overnight incubations, the cells were treated with or without different concentrations of 3b for 96 h. Cells were then labeled with Annexin V and PI as previously described (Ibba et al., 2021 (link)). Stained cells were then analyzed by flow cytometry, measuring the fluorescence emission at 530 and 620 nm using 488 nm excitation laser (MoFloAstrios EQ, Beckman Coulter). Cell apoptosis was analyzed using Software Summit Version 6.3.1.1, Beckman Coulter.