Figure 5.

Tests of cell division, colony formation and oncogenic effects of mutant GLI1 proteins. (A) NIH3T3 cells were infected with vector, GLI1^WT, GLI1^3A, or GLI1^3E lentivirus, with Flag tags on each protein, and selected for 7 days with puromycin (2.5ugml^-1). Cell lysates were analyzed by immunoblotting with Flag antibody to measure the amounts of the expressed proteins. (B) This experiment used NIH3T3 GLI1-stable cell lines (vector control, WT, 3A, and 3E). 5 × 10³ cells were seeded into 12-well plates for growth assays, each cell type in triplicate, and cells were counted using a hemocytometer for three consecutive days. The experiment was repeated three times (“ * ” p<0.01, “ ** ” p<0.001). (C) NIH3T3 cells with GLI1^WT, GLI1^3A, or GLI1^3Estably expressed were seeded into 6-well plates for colony formation assays for 2 weeks. Colonies larger than 1.5 mm were counted. (D) As in (C) the cells were treated with 2DG (25mM) and 2DG-containing medium. The medium in 2DG-treated wells was changed every three days to refresh the 2DG. Colony numbers were counted two weeks later. In (C) and (D), each cell line was seeded in duplicate, with N=3 (“ * ” p<0.05, “ *** ” p<0.0001). (E) NIH3T3 GLI1-stable cell lines (Vector control, WT, 3A, and 3E). 10⁷ cells were injected subcutaneously into the nude mice and tumor growth was monitored for three weeks (“ ** ” p<0.001).