Response to SMO antagonist PF-04449913 varies among AML cell lines. (A) AML cell lines were treated with 100 nM of PF-04449913 for 48 hours. Cell viability was tested by trypan blue exclusion assay. *P < .05 vs vehicle-treated cells. Treatment with PF-04449913 decreased cell viability in KG1 cells with no change in K562 and KG1a cells. (B) GLI1 transcript levels, which are an indicator of Hh pathway activity, were measured by Q-PCR. GLI1 transcript levels were reduced in KG1 cells only with PF-04449913 treatment. (C) AML cell lines (KG1, KG1a, and K562) were transiently transfected with a GLI-responsive luciferase reporter or pGL3-basic, a plasmid lacking GLI binding site, as a negative control. Relative luciferase levels were measured at 48 hours posttransfection. (D) SMO protein levels were measured in cell lines treated with PF-04449913. (E) K562 and KG1 cells were transiently transfected with SMO siRNA, respectively. Non-specific scrambled siRNA (NS siRNA) was used as control. Cell viability was tested by trypan blue exclusion assay. Transfection efficiency of SMO siRNA transfection efficiency was measured by western blotting for SMO protein levels. GLI1 and GLI3 protein levels were also measured by western blotting. (F) K562 cells were transiently transfected with GLI1 siRNA. NS scrambled siRNA was used as control. Cell viability was tested by trypan blue exclusion assay. *P < .05 vs NS siRNA control. Transfection efficiency of GLI1 siRNA was measured by western blotting using GLI1 antibody. Glyceraldehyde-3-phosphate dehydrogenase was used as a loading control in panels D-F.