GLI3 is downregulated in AML and GLI3R is a critical regulator of Hh signaling in AML. (A) Western blotting was used to measure GLI3 protein levels in clinical specimens from patients with AML and normal bone marrow (NBM). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Compared with NBM, AML samples showed significantly lower GLI3 levels. (B) AML cell lines and primary AML blasts were treated with PF-04449913, and cell lysates were analyzed for GLI1 and GLI3 protein levels. SMO antagonist increased the GLI3R:GLI3F ratio in responsive KG1 cells and primary AML11 blasts with no change in nonresponsive K562 and KG1a cells and primary AML2 blasts. PF-04449913 decreased GLI1 levels in KG1 and AML11 cells as shown by GLI1 densitometry. GAPDH was used as a loading control in all experiments. Densitometry analysis was done by using Image Studio Lite software. (C) K562 cells were transiently transfected with empty and Myc-tagged GLI3R constructs (0, 0.25, 0.5, 0.75, and 1 μg) followed by PF-04449913 treatment. Forced expression of GLI3R at 0.5 μg significantly reduced cell viability, which was additionally reduced after PF-04449913 treatment. *P < .05. (D) After cells were transfected with the indicated constructs and treated with PF-04449913, cell lysates were prepared and subjected to immunoblotting with anti-GLI1, anti-Myc, and anti-GAPDH antibodies. (E-F) K562 cells were transiently transfected with iLenti-si-Gli3 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 GLI3 siRNA was measured by western blotting by using GLI3 antibody. (G- H) KG1 cells were transfected with iLenti-si-Gli3 siRNA followed by treatment with PF-04449913. NS scrambled siRNA was used as control. Cell viability was tested by trypan blue exclusion assay. *P < .05. GLI1 and GLI3 protein levels were measured by western blotting. GAPDH was used as a loading control.