Figure 2
GLI1 expression in SUM149 cells is Hh-ligand and SMO independent. (A) RT–PCR analysis of GLI1 mRNA expression in SUM149 and rSUM149 cells treated for 72 h with media or 2 μg ml–1 recombinant ShhN (left panel) or with 100 μg ml–1 5E1 antibody or 100 μg ml–1 isotype control antibody (right panel). β-Actin was used as an internal control. (B) Immunoblot analysis for GLI1 protein expression in SUM149 cells (7 × 104 cells per well in six-well plates) treated for 72 h with media or ShhN (left panel) or with 5E1 antibody or control antibody (right panel). Membranes were stripped and reprobed for β-actin as an internal control for equal loading. (C) Cell proliferation was determined by MTT assay in SUM149 cells (2000 cells per well in 96-well plates) treated for 72 h with media or ShhN (left panel) or with 5E1 antibody or control antibody (right panel). (D) RT–PCR analysis of GLI1 mRNA expression in SUM149 (upper panel) and rSUM149 (lower panel) cells treated for 72 h with the indicated concentrations of KAAD-cyclopamine (KAAD-cyc) or its inactive analogue tomatidine. β-Actin was used as an internal control. (E) Cell proliferation was determined by MTT assay in SUM149 cells (2000 cells per well) plated in 96-well plates following treatment for 72 h with the indicated concentrations of KAAD-cyc and its inactive analogue tomatidine.
