Fig. 2: SNS-032 induces apoptosis in ESCC cells.

A, B ESCC cells were incubated with increasing concentrations of SNS-032 for 48 h; the apoptotic cells were detected using flow cytometry after dual staining with annexin V-FITC and PI. A Representative flow cytometric dot plots for KYSE30 cells are shown; B quantitative analysis of dead cells from five independent experiments. Dead cells were the sum of cells with single or dual stained by annexin V-FITC or PI. C Dose-dependent cleavage of PARP and Caspase-3 was detected by western blot analysis in ESCC cells incubated with SNS-032 for 48 h. D, E SNS-032 triggered loss of mitochondrial membrane potential in ESCC cells. ESCC cells (KYSE30, KYSE70, KYSE450, and KYSE510) were treated with or without 0.25 μM SNS-032 for the indicated durations, stained with JC-1, and subsequently detected by flow cytometry. D Representative flow cytometric dot plots for KYSE30 cells are shown; E quantitative analyses from five independent experiments are shown. *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA with post hoc intergroup comparison by Tukey’s test. F SNS-032 induced the release of Cytochrome c and AIF from mitochondrial into cytosol in ESCC cells. Mito (mitochondria): the positive control; COX IV served as a mitochondrial indicator to rule out contamination of cytosolic fractions from mitochondria. Actin was used as an internal control.