Igf2 overexpression does not initiate adrenal tumourigenesis. A- β-catenin is not activated in AdIgf2 adrenals.

<p>β-catenin expression was analysed by immunohistochemistry in wild-type (WT, a), ΔCat (positive control, b) and AdIgf2 (c–d) adrenals. Section in c was counterstained with haemotxylin. Nucleo-cytoplasmic staining of β-catenin (white arrowheads) is restricted to zona glomerulosa in WT and AdIgf2 adrenals but spreads throughout the cortex and inside the medulla in ΔCat adrenals. Black arrows show infiltrating mesenchymal subcapsular cells. <b>B -Wnt pathway is not activated in AdIgf2 adrenals.</b> Expression levels of Axin2 and Lef-1, two canonical Wnt pathway targets, were determined by RTqPCR with cDNAs from wild-type, AdIgf2 and ΔCat (positive control) adrenals. Bars represent the mean relative quantification (Rq Tg/WT) of gene expression for each gene in at least 6 adrenals per genotype ± standard deviation. <i>P</i>-value was calculated using Student's <i>t</i>-test. <b>C- Analysis of Ki67 expression.</b> Ki67 was detected by immunohistochemistry on wild-type (WT, a) and AdIgf2 adrenals (b). <b>D- </b><b>Proliferation is not increased in AdIgf2 adrenals.</b> Numbers of Ki67-positive cells in a whole adrenal section were counted separately in the cortex (Co) and medulla (M). For each zone, the number of cells was corrected for surface and is expressed as a percentage of Ki67-positive cells in one of the control individuals. Bars represent the mean of at least 7 individual counts in wild-type and AdIgf2 adrenals ± standard deviation. <i>P</i>-value was calculated using Student's <i>t</i>-test. <b>E- Cyclin D1 expression is not increased in AdIgf2 adrenals.</b> Expression of Cyclin D1 was analysed by RTqPCR with cDNAs from Wild-type and AdIgf2 adrenals. Bars represent the mean relative quantification (Rq AdIgf2/WT) of gene expression in at least 5 adrenals per genotype ± standard deviation. <i>P</i>-value was calculated using Student's <i>t</i>-test. <b>F- Igf2 overexpression does not induce adrenal tumour formation.</b> The phenotype of AdIgf2 adrenals was followed over a 14 months time course by haematoxylin & eosin staining (b–c) and immunohistochemistry for Ki67 (e–f). A 10 month-old wild-type adrenal was included as a reference (WT, a, d). Some twelve month-old AdIgf2 mice were treated with ACTH for two months in order to increase Igf2 expression. Adrenal histology and proliferation were then assessed by haematoxylin & eosin staining (g) and Ki67 immunohistochemistry (h). Arrows show mesenchymal subcapsular cells. M, medulla; F, fasciculata; G, glomerulosa; Co, cortex. Scale bar is 80 µm.