Figure 3

Up-stream signaling pathways affecting GL-mediated ATF3 activation. (A, B) Luciferase construct containing -1420 to +34 of human ATF3 promoter region was cotransfected with pRL-null vector. Then, the cells were pretreated with 20 μM of PD98059 (ERK1/2 inhibitor) or SB203580 (p38 inhibitor) and then cotreated with 100 μg/ml of GL for 24 h. *P <0.05 compared to cells without the treatment of inhibitors. (C, D) HCT116 and SW480 cells were treated with 100 μg/ml of GL for indicated times. Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibodies against p-ERK1/2 and ERK1/2. Actin was used as internal control. (E) HCT116 cells were pre-treated with 20 μM of PD98059 (ERK1/2 inhibitor) or SB203580 (p38 inhibitor) and then co-treated with 100 μg/ml of EAFAD-B for 6 h. Cell lysates were subjected to SDS-PAGE the Western blot was performed using antibodies against ATF3. Actin was used as internal control. (F) HCT116 cells were pretreated with 20 μM of PD98059 (ERK1/2 inhibitor) for 2 h and then co-treated with 100 μM of GL 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.