Figure S1. Expression of LHβ hormone subunit in pituitary glands of young rats. (AC) Pituitary glands from 4-month-old wild-type rats. (DF) Pituitary glands from a 4-month-old mutant rat. Immunohistochemistry was performed using anti-LH antibody and counterstained with haematoxylin. In the mutant rat pituitary discrete nodules of LH-positive cells can be observed, while the wild-type glands show a sparse distribution of LH-positive cells. Scale bars: 200 μm (A,D); 100 μm (B,C,E,F). Original magnification: 4× (A,D); 10× (B,C,E,F).

Figure S2. Hormone profiling of the rat pituitary lesions. (A) Summary of the percentage of lesions showing immunoreactivity for the indicated hormones. Ten mutant rat pituitaries were subjected to immunohistochemistry with antibodies directed against the seven hormones and counterstained with haematoxylin. In total, 55 individual lesions were scored. (B,C) Representative stainings of the few lesions we scored positive for GH (B) or PRL (C). Scale bars: 100 μm. Original magnification: 10×.

Figure S3. Expression of lineage-specific transcription factors SF1, Tpit and Pit1 in pituitary tumours from adult mutant rats. (A–D) Immunohistochemistry was performed using antibodies against SF1 (A), Tpit (B) and Pit-1 (C,D) and counterstained with haematoxylin. The tumour areas are indicated (T). Panel D shows a lesion that was also positive for PRL. Original magnification: 10×. Insets: 20×.

Figure S4. Expression of αGSU, GH, PRL and Ki67 in pituitary glands of 4-month-old mutant rats. Immunofluorescent staining with the indicated specific primary antibodies was performed on rat formalin-fixed, paraffin-embedded (FFPE) tissues. Nuclei were counterstained with DAPI. Tumour lesions (panels E–H, M–P, U–Y) and normal adjacent areas (panels A–D, I–L, and Q–T) within the same gland are shown. The lesions are circled in white. Cells showing the colocalization of GH and αGSU (panel H) or the colocalization of PRL and αGSU (panel P) are indicated with white arrowheads. The few Ki67-positive cells in nontumorous tissue in panels S and T are indicated with white arrowheads, while the αGSU-positive cells in panels R and T are indicated with white arrows. In the tumour area (panels U, Y) many cells show the characteristic punctuate nuclear Ki67 staining, indicating sustained proliferation rates. Scale bars: 100 μm. Inset in panel P, 40× .

Figure S5. Gene expression in rat pituitary tissues and primary pituitary tumour cell cultures. (A) Quantitative RT-PCR for Cdkn2b (p15), Cdkn2a (p16), Cdkn2c (p18), Cdkn1a (p21) and Cdkn1b (p27). RNA extracted from four normal pituitaries (empty bars) and four pituitary tumour tissues (filled bars) was used. Expression values were normalized against the average of the wild-type tissues arbitrarily set to 1. Bars, standard deviation. (B) Primary cultures were established from the pituitary tumours of four mutant rats (No. 1–4) at the age of 6 months. Semi-quantitative RT-PCR for the genes encoding the indicated proteins was performed. Actin was used as internal control for RNA quantity and quality. GH3 cells acted as control as they do not express FSHβ and LHβ. C, negative control.

Table S1. Antibodies used, their dilution and source.

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