YK-135 induces AMPK-mediated apoptosis in EMT-subtype gastric cancer cell lines. (A) YK-135-induced AMPK signaling was assessed by western blotting of whole-cell lysates from the indicated EMT and non-EMT cell lines. (B) Evaluation of cell cycle arrest by propidium iodide staining and flow cytometry analysis after YK-135 treatment for 24 h. Ten thousand cells were counted per analysis. Data represent mean ± SD from experiments in triplicate. P-values were determined by two-way ANOVA (*P < 0.05, **P < 0.01, n.s.: not significant). (C) Flow cytometry analysis of MitoTrackerTM Red CMXRos and MitoTrackerTM Green FM-stained cells to detect changes in mitochondrial membrane potential (ΔΨm) and mitochondrial mass induced by YK-135. (D) Representative flow cytometry histogram of MitoSOX staining in DMSO (light red), 5 μM YK-135 (light blue), and 10 μM YK-135 (orange). (E) Representative scatter plots of apoptosis analysis using Annexin V-FITC/PI dual staining and flow cytometry after YK-135 (5 μM) treatment for 48 and 72 h. (F) YK-135 induced apoptosis was assessed by western blotting using anti-cleaved PARP antibody in SNU484 and MKN45 cells post-incubation for indicated time points.
