Fig. 4.

Screening of human GBM operative specimens for alterations in expression and mutations. (A) RT-PCR screening of a panel of 22 human GBM operative specimens. GATA6 expression was absent in 20/22 samples tested. GAPDH was used as a positive control marker. (B) Chromatograms of homozygous frameshift mutations identified in the GATA6 DNA binding domain and C terminus of five GBM specimens. These specimens also had loss of GATA6 RNA expression by RT-PCR (A), with loss of GATA6 protein expression in 19 specimens available for IHC evaluation (Fig. 5A). (C) Schemata of the human GATA6 protein with locations of homozygous frameshift mutations identified in the DNA binding domain (3/5) and C terminus (2/5) of GBM specimens and associated with LOH (SI Fig. 10). (D) DNA binding assays of GATA6 patient GBM mutations using the p450-Cytochrome:C17 promoter:pGL3 firefly luciferase reporter in U87 cells. Negative controls included nontransfected U87 plus transfection with the pcDNA3.1+ vector lacking only the GATA6 gene. The data (expressed as mean ± SEM) demonstrate that wild-type GATA6 induced significant expression (P < 0.001) of the firefly luciferase reporter from the p450-Cytochrome:C17 promoter. However, the GATA6 frameshift mutations identified from the GBM specimens resulted in minimal nonsignificant induction of the firefly luciferase reporter from the p450-Cytochrome:C17 promoter.