Fig 1: Apoptotic effects of TSN on MKN-45 cells. (A) MKN-45 cells were seeded into 6-well plates and treated with varying concentrations of TSN for 48 h, incubated with acridine orange, and visualised using laser confocal microscope. (B and C) Early apoptotic effects of TSN were evaluated by flow cytometry analysis after staining with annexin V-FITC and PI. (D) The mRNA expression of Caspase-3, 8, 9, BAX, BCL2, FAS, Cyt c and APAF1 was detected using reverse transcription quantitative PCR in TSN-treated MKN-45 cells. (E and F) Protein expression levels of Caspase-3, 8, 9, Bax, BCL2, Fas, Cyt c and APAF-1 were detected using western blotting in TSN-treated MKN-45 cells. ß-actin was used as an internal control. All results are expressed as the mean ± SEM of three independent experiments; n=3; *P<0.05, **P<0.01, ***P<0.001 vs. 0 nM. Cyt c, cytochrome c; TSN, toosendanin; APAF1, apoptotic protease activating factor 1; 5-FU, fluorouracil.