A. The morphologic changes of CAL27 treated with NSC74859 were captured using fluorescence microscopy with Hoechst 33258 staining. Scale bar 20 μm; B. CAL27 cells were treated with 50 μM, 100 μM, and 200 μM of NSC74859 for 24 h and stained with Annexin V/PI, then analyzed by flow cytometry. The percentages of Annexin V-positive cells were presented in bar charts; C. CAL27 cells were incubated with 100 μM of NSC74859 for 6, 12 and 24 h, then analyzed by flow cytometry. The percentages of Annexin V-positive cells were presented in bar charts; **P < 0.01. One-way ANOVA with post-Dunett analysis was used by GraphPad Prism5; D. CAL27 cells were treated with different concentration of NSC74859 for 24 h then western blot analysis was performed to assess the expression level of STAT3 and p-STAT3T705, cleaved-PARP (Cl-PARP) and cleaved-caspase 3(Cl-casp 3), GAPDH served as a loading control; Relative density data were calculated by Image J, and the data represented mean of three independent experiments. *P < 0.05, **P < 0.01; E. CAL27 cells were treated with 100 μM of NSC74859 for 6, 12 and 24 h then western blot analysis was performed to assess the expression level of STAT3 and p-STAT3T705, Cl-PARP and Cl-casp 3, GAPDH served as a loading control; Relative density data were calculated by Image J, and the data represented mean of three independent experiments. *P < 0.05, **P < 0.01, One-way ANOVA with post-Dunett analysis was used by GraphPad Prism5.