Effect of EAFAD-B on ATF3 expression and ATF3-mediated apoptosis in human colorectal cancer cells. (A, B, I, J) HCT116 and SW480 cells were plated and then treated with EAFAD-B. Cell lysates were subjected to SDS-PAGE the Western blot was performed using antibodies against ATF3. Actin was used as internal control. (C, D) RT-PCR analysis of ATF3 gene expression, total RNA was prepared after EAFAD-B treatment for 24 h. GAPDH was used as internal control (E, F, G, H) For ATF3 promoter activity, luciferase construct containing -1420 to +34 of human ATF3 promoter region was cotransfected with pRL-null vector and the cells were treated with EAFAD-B and luciferase activity was measured. *P < 0.05 compared to cells without EAFAD-B treatment. (K) ATF3 siRNA was transfected into HCT116 for 48 h and then EAFAD-B was treated for 24 h. Cell lysates were subjected to SDS-PAGE the Western blot was performed using antibodies against PARP. Actin was used as internal control.