Figure 1
Hck is a direct Shh/Gli1 target gene. (a) NIH3T3 cells were treated with or without Shh-conditioned media. Levels of Hck and Gli1 mRNAs were determined by RT–qPCR. (b) Hck and Gli1 mRNA levels were determined by RT–qPCR in CGNP cells treated with or without Shh-conditioned media. (c) NIH3T3 cells were infected with empty vector or lentiviruses for expression of Gli1 proteins. Levels of Hck and Shh target gene Ptch1 were determined. (d) Levels of Hck were determined by RT–qPCR in NIH3T3 cells infected with lentiviruses expressing different Gli proteins. (e) A schematic map of the 1.7 kb Hck regulatory region used for ChIP and reporter assays. The Gli binding site (indicated by the arrow) and the transcription start site (TSS) are shown. Small bars indicate the location of ChIP PCR products. (f) NIH3T3 cells were infected with empty vector control or HA-Gli1 expressing lentiviruses. ChIP-qPCR analyses were performed with anti-HA antibody. A region in CD4 was used as a negative control. (g) The 1.7 kb wild-type full-length Hck enhancer region (−1575; E: EcoRI site. X: XhoI site) and mutants including a truncated construct (−701), a construct with the putative Gli1 binding site deleted (−1575D) or mutated (−1575 m) were cloned upstream of a luciferase reporter gene. Plasmids expressing control (empty vector) or Gli1 proteins were co-transfected with reporters into NIH3T3 cells. Relative luciferase activities are shown on the right. Presented are means plus s.d. Statistical analyses were performed using the Student’s t-test; **P<0.01. qPCR, quantitative PCR.
