Fig. 5

Calcium and phosphate determinations under different calcifying conditions in HAoSMCs. (A) (B) Extracellular and intracellular calcium concentrations (µg calcium/µg protein), (C) (D) extracellular and intracellular phosphate (nmol phosphate/µg protein). All determinations were measured in mock and HAoSMCs overexpressing sclerostin, under non-calcifying and calcifying conditions. (E) Representative microphotographs obtained at 5X magnification of Alizarin Red staining of mock and HAoSMCs overexpressing sclerostin, under both non-calcifying (top) and calcifying (bottom) conditions and normalized by total protein content. Scalebars: 200 μm. Calcium mineral depositions were analyzed in the different conditions and results were expressed as the OD 405 nm/µg protein; n = 3 biological replicates/group and n = 3 technical replicates/ biological replicates were performed. Data are represented as the mean ± standard deviation. The p-values between groups were determined by the unpaired Student´s t-test. In the figure A and C * = p < 0.05 vs. non-calcifying conditions. In figure B, * = p < 0.05 vs. calcified mock. HAoSMCs, Primary Human Aortic Smooth Muscle Cells; OD, optical density.