GLI3R suppresses Hh signaling via transcriptional repression of AKT. (A) K562 cells were transiently transfected with the GLI3R expression construct. After 48 hours, cell lysates were separated and immunoblotted using anti-AKT, anti-ERK1/2, anti-Myc, and anti-glyceraldehyde-3-phosphate dehydrogenase antibodies. Total AKT levels were significantly downregulated in GLI3R-expressing K562 cells. (B-C) Expression of AKT in GLI3R-overexpressing cells rescues the GLI3R-mediated growth inhibition. K562 cells were transiently transfected with the GLI3R and/or the AKT1 expression construct. Cell viability was tested by using trypan blue exclusion assay. *P < .05 compared with control. (D) Schematic diagram of the AKT1 promoter region with potential GLI3 binding site (BS). The 9 bp GLI-binding site sequence is shown. (E) Fold enrichment of GLI3 on AKT. ChIP assay was performed with control immunoglobulin G and GLI3R-specific antibodies.