Arterial calcifications and increased expression of vitamin D receptor targets in mice lacking TIF1α

Fig. 4. The TIF1a protein is detected in a large number of epithelia and the uterine stroma. Immunolocalization of TIF1a using a specific mAb (red nuclear signals). (A, C, D, E, G, I, J, and K ) Tissues from WT mice. (B, F, H, and L) Tissues from TIFa^-/- mutants, serving as negative controls of the immunostaining procedure. The right side of each panel is a Photoshop-generated superimposition of the immunofluorescent signal shown in A (red), the DAPI nuclear counterstain (blue), and the tissue autofluorescence (green). CC, colonic crypts; EnP and ExP, endocrine and exocrine pancreas, respectively; EO, epithelium of the oviduct; F, ovarian follicle; G, uterine glands; L, epididymal lumen; SE, seminiferous epithelium; ST, uterine stroma. Note that the signal in the lumen of some blood vessels (asterisks) is unspecific and reflects the binding of the secondary anti-mouse Cy3-conjugated IgG to circulating immunoglobulins. (Scale bar: A, B, D, I, J, K, and L, 80 mm; C, E, F, G, and H, 25 mm.)

Fig. 5. Examples of intranuclear inclusions in hepatocytes of 3-month-old TIF1a^-/- mutants. (A and B) Hematoxylin/eosin stain. (C) Electron-microscopic image. Intranuclear inclusions (yellow arrowheads) are invaginations of the cytoplasm (CY) into the nucleus (N). Note that at this age livers of TIF1a^-/- mutants do not yet display signs of neoplastic transformation. (Scale bar: A and B, 40 mm; C, 1 mm.)

Table 3. Hematological parameters in WT (TIF1a^+/+) and TIF1a^-/- mice (means ± SD) at the age of 8 months

n = 5 mice of each genotype and gender. MCH, mean corpuscular hemoglobin; MCHC, mean cell hemoglobin concentration.

Table 4. Primer sequences

SI Text

Alizarin Red Staining of Mouse Skeletal Preparations

The mice were killed. The skin of the trunk, head, and limbs was removed. The ventral abdominal wall was incised. With forceps all thoracic and abdominal viscera were removed through this incision. The viscera were fixedx overnight in 95% ethanol containing 1% acetic acid (vol/vol) at room temperature. Soft tissues were cleared in 2% KOH for 12 h with two changes or, alternatively, in 1% KOH for 48 h. Bone was stained in alizarin red solution (alizarin red sodium sulfonate; CI: 58 005; Sigma; 75 mg alizarin red in 1 liter of 1% KOH) overnight; the solution can be kept for several weeks at room temperature. It was distained in 20% glycerol, 1% KOH for 7 days, and the solution was changed every day. The remaining soft tissues was dissected away if required and transferred in 20% glycerol, 20% ethanol for 1 week. Then it was transfer in 20% glycerol, 50% ethanol overnight then to 100% glycerol for storage.

Immunohistochemistry