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. 2017 Jul 25;8:536. doi: 10.3389/fphys.2017.00536

Figure 4.

Figure 4

BAV- and TAV-derived smooth muscle cells (SMC) differ in their differentiation properties in osteogenic conditions. SMC from patients with thoracic aortic aneurysm with either tricuspid aortic valve (TAV) or bicuspid aortic valve (BAV) and controls (C) were cultured with control or osteogenic [in the presence of 10 mM β-glycerolphosphate, 200 μM L-ascorbic acid and 100 nM dexamethasone (Sigma)] medium for 5 days. At day 5, total RNA and protein were extracted for qPCR analysis (A) and Westernblotting (B), respectively. TAV, n = 8, BAV, n = 7, C, n = 8. (A) Culture in osteogenic conditions was accompanied by SMC-marker gene activation, which is stronger in aneurysm-derived cells, and also by statistically significant increase in RUNX2 expression only in BAV-derived cells. Relative mRNA levels for ACTA2, TAGLN, and RUNX2 in human aortic SMC were determined by qPCR and normalized to GAPDH. Groups were compared using Mann-Whitney nonparametric test; line represents the median; *p < 0.05. (B) In vitro induced calcification. SMC were cultured in osteogenic conditions, and ALP activity was visualized with BCIP/NBT at day 14. (C) Representative Western blots for detection of smooth muscle actin (SMA), and SM22α, in human aortic SMC after culture in osteogenic conditions. α-Tubulin was used as a control to verify total amount of protein.