Fig. 3. Effect of calcitriol and paricalcitol on the canonical Wnt/ -catenin signaling pathway during HASMC calcification. HASMCs are incubated for 24 h in a high (3.3 mmol/l) phosphate (HP) medium (calcification medium) alone or supplemented with either calcitriol 10 8 M (HP CTR) or paricalcitol 3·10 8 M (HP PC). Cells incubated in normal phosphate (0.9 mmol/l) medium are used as controls. A: levels of nuclear -catenin is assessed by western blotting of nuclear extracts. Image represents 3 different experiments. Quantification is performed by measurement of the integrated optical density (OD) and normalized to TFIIB levels. Data are means SE of the 3 independent experiments. *P 0.05 vs. control. #P 0.05 vs. HP. B: HASMCs are incubated for 24 h in a high (3.3 mmol/l) phosphate (HP) medium (calcification medium) alone or supplemented with either calcitriol 10 8 M (HP CTR) or paricalcitol 3·10 8 M (HP PC) alone or with the addition to culture medium (100 ng/ml) of commercially available Dickkopf-related protein 1 (DKK-1), a specific endogenous extracellular antagonist of the Wnt signaling. Intracellular localization of -catenin is visualized by immunofluorescence using confocal microscopy. For each treatment, -catenin staining (green immunofluorescence) is shown at left; at middle, the same sample is counterstained with DAPI (blue) for nuclear stain; the merged image is shown at right. Images represent 3 different experiments. C: quantification of nuclear -catenin staining is performed by the Mander’s coefficient (M2 plugin: DAPI vs. green). *P 0.05 vs. control. #P 0.05 vs. the same treatment without DKK-1.
...read more