Figure 4

Up-stream signaling pathways affecting EAFAD-B-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), SB203580 (p38 inhibitor) or SB216763 (GSK3β inhibitor) and then co-treated with 200 μg/ml of EAFAD-B for 24 h. Luciferase activity was measured. *P < 0.05 compared to cells without EAFAD-B treatment. (C, D) HCT116 and SW480 cells were pre-treated with 20 μM of PD98059 (ERK1/2 inhibitor), SB203580 (p38 inhibitor) or SB216763 (GSK3β inhibitor) and then co-treated with 200 μg/ml of EAFAD-B for 24 h. Cell lysates were subjected to SDS-PAGE the Western blot was performed using antibodies against ATF3. Actin was used as internal control. (E) SW480 cells were treated with 200 μg/ml of EAFAD-B for indicated times. Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibodies against p-p38, p38, p-ERK1/2, ERK1/2, p-GSK3β or GSK3β. (F) HCT116 cells were pre-treated with 200 μg/ml of EAFAD-B for 12 h and then co-treated with CHX (10 μg/ml) for the indicated times in absence/presence of EFAD-B. Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibodies against ATF3. Actin was used as internal control.