Fig. 6

DMH2 regulation of cell death involves the downregulation of XIAP, which regulates the activity of TAK1. a-d Western blot analysis of cells treated with DMH2, DMH1, or LDN and in combination with SB for 3 days. Only DMH2 induced the downregulation of XIAP. (e) The knockdown of XIAP was performed using siRNA in H1299 cells and after 3 days percentage of dead cells determined. (f) Western blot analysis showing that knockdown of XIAP decreases the expression of pTAK1. (g) H1299 cells were transiently transfected with mutant XIAP with ubiquitination sites removed. Western blot analysis after 48 h demonstrates increased expression of pTAK1. (h) H1299 cells were transiently transfected with control vector and mutant XIAP containing a N-terminal c-Myc epitope. Cells immediately after transfection were treated with DMSO or DMH2 for 3 days. Western blot demonstrating that DMH2 decreased expression of wild type XIAP but not of mutant XIAP. (i) H1299 cells were stably transfected with vector control and mutant XIAP. Cells were treated with and without DMH2 for 3 days and the percentage of cell death was determined. DMH2 only induced cell death in the vector control cells. All experiments were performed at least 3 times. (j) Western blot analysis of stable transfected cells treated with DMSO or DMH2 for 3 days. The bottom panel shows a shorter exposure of the panel above (n = 2)