Fig. 2
Northern analysis, immunohistochemistry, and in situ hybridization of adrenal tissue from normal and transgenic animals. (A) Normal adrenals are devoid of GATA-4 mRNA, but adrenal tumor tissue from transgenic animals expresses abundant GATA-4 mRNA; GATA-4 mRNA is strongly upregulated also in the cell line originating from these tumors. (B) In contrast, GATA-6 mRNA is detected in normal adrenals but not in adrenal tumors or adrenal tumor cell line. (A, B) Normal mouse testis was used as a positive control and spleen as a negative control for GATA-4 mRNA expression. EtBr, ethidium bromide, Sp, spleen; Te, testis; Adr. Co., normal adrenal; Calphal cells, murine adrenal tumor cells; M, male; F, female. The bars in A&B show the mean ± SEM calculated from three independent experiments performed in duplicate. *, p < 0.05; **, p < 0.001 for GATA-4 and GATA-6 mRNA expression after the indicated treatment vs. that in the spleen (control). (C) GATA-4 mRNA (white column) is absent and GATA-6 (black column) present in the adrenals of normal animals, of normal gonadectomized animals, and of transgenic animals. Whereas GATA-4 is present and GATA-6 absent in the adrenals of gonadectomized transgenic animals, i.e. in adrenal tumors. The upper panel shows representative 28S ribosomal RNA band in the gel stained with EtBr, and Northern blots for GATA-4 and GATA-6. TG Adrenals, the adrenals of the transgenic mice; F, female; M, male. (D-F) Immunoperoxidase staining shows the presence of GATA-4 protein in the adrenal tumor tissue (tu, arrowheads). A small area of normal adrenal tissue can be seen on the edge of the tissue sample (nl, arrows); (D) H&E staining of the tumor; (E) immunoperoxidase staining for GATA-4; (F) control staining with nonimmune serum. Original magnification (×100), bar =100 µm. (G-I) In situ hybridization demonstrates the presence of GATA-4 mRNA (H) in adrenal tumor tissue, whereas it is essentially devoid of GATA-6 mRNA (I); (G) H&E staining of adjacent section. Original magnification (×100), bar = 100 µm.