Effects of GL on ATF3 activation in human colorectal cancer cells. (A, B) HCT116 and SW480 cells were treated with 0, 25, 50 and 100 μg/ml of GL for 24 h. Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibodies against ATF3. Actin was used as internal control. For RT-PCR analysis of ATF3 gene expression, total RNA was prepared after GL treatment for 24 h in dose-course experiments (C, D) or for the indicated times in time-course experiments (G). GAPDH were used as internal control. 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 GL for 24 h in dose-course experiments (E, F) and luciferase activity was measured. *P <0.05 compared to cells without GL treatment. (H) ATF3 siRNA was transfected into HCT116 for 48 h and then GL 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.