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. 2007 Apr 26;104(19):8053–8058. doi: 10.1073/pnas.0611669104

Fig. 6.

Fig. 6.

Functional studies of human GATA6 shRNA knockdown and reexpression. (Ai) GATA6 expression was knocked down by two shRNAs (human exons 4 and 5) from a stably integrated pSIREN-RetroQ vector. Parental astrocytes and those expressing control shRNA were used as negative controls. (Aii) MTT proliferation assay on hTERT immortalized nontransformed NHA and NHA:V12Ha-Ras human astrocytes. GATA6 knockdown did not induce a significant in vitro proliferation advantage in the NHA or NHA:V12Ha-Ras cells (P > 0.05). (B) Intracranial injection into Nod-Scid mice with 106 NHA:V12Ha-Ras + GATA6 shRNA knocked-down astrocytes, but not controls, resulted in in vivo malignant astrocytomas (H&E; Magnification: ×200), demonstrating tumor cells with nuclear atypia and mitosis. (C) GATA6 reexpression induced antiproliferative effects on U87:Tet ON-GATA6 lines treated with Dox (P < 0.05). Leaky expression of GATA6 in the pREV-TRE vector can account for slight inhibition of proliferation even in untreated cells compared with parental U87 cells over the 6-day analysis period. (D) Cell cycle flow cytometry analysis of U87 cells transiently expressing GATA6 under control of the cytomegalovirus promoter (pcDNA3.1+). Nontransfected and empty vector (pcDNA3.1+) transient transfected U87 cells served as negative controls. Transient reexpression of GATA6 induced a significant (P < 0.05) increase in G1 arrested cells compared with the negative controls. (E) Intracranial xenografts with 106 U87:Tet ON-GATA6 cells with or without Dox in the drinking water. Tumor growth was not seen after ≈4 months in mice treated with Dox (Left; arrow shows injection tract). In contrast, mice without Dox had to be killed at 4 weeks, with development of large tumor nodules at the site of injection (Center; Right shows a magnified view of the tumor). (Magnification: ×40 in Left and Center and ×400 in Right. Scale bars: 250 μm in Left and Center and 25 μm in Right.) (F) GATA6 regulation of VEGF expression. U87 cells express VEGF (similar to control mouse brain endothelial cells) but not GATA6. Transient reexpression of GATA6 in the U87 cells inhibits VEGF expression.