DTCD affects the levels of MAPK phosphorylation.

<p>A2780 cells were incubated with 10 µM of DTCD for indicated times (A) or with indicated amounts of DTCD+TRAIL for 1 h (B) and the levels of -ERK, -JNK, - p38 and their phosphorylated forms were determined by western blotting. (C) MAPKs are involved in DTCD-mediated upregulation of Sp1 DNA binding activity. A2780 cells were stimulated with 10 µM DTCD for 24 h after pretreatment with 20 µmol/L PD98059, 20 µmol/L SP600125, and 10 µmol/L SB203580 for 1 h. Then, Sp1 DNA binding activity and nuclear translocation were analyzed by EMSA (<i>left</i>) and Western blotting (<i>right</i>), respectively. (D) Effects of PD98059 on DR5 promoter activity and expression assay. PD98059 inhibits DR5 luciferase activity induced by DTCD. pDR5/605 plasmid was transfected into A2780 cells, which were then pretreated with 20 µmol/L PD98059 for 1 h and further treated with 10 µM DTCD for 24 h, lysed and assayed for luciferase activity and Western blot assay, respectively. (E) Effects of PD98059 on the cytotoxicity induced by DTCD and TRAIL.A2780 cells were treated with DTCD (10 µM) and TRAIL (200 ng/mL) in the presence of indicated amount of PD98059 for 24 h. Cell viability was determined by MTT assay. (F) Cells were transfected with ERK1 siRNA, ERK 2 siRNA or control siRNA, respectively. 24 h after the transfection, cells were treated with 10 µM DTCD for another 24 h, and cell extracts were subjected to Western blotting. (G) Cells were transfected with JNK1/2 siRNA or p38 siRNA, then treated with 10 µM DTCD for 24 h. Western blotting was then used to analyze the extracts for JNK, and p38 MAPK expression.