FIGURE 4.

Inhibition of AKT‐ and ERK signalling attenuate EV^UR‐mediated effects on VSMC calcification. A, VSMC were cultured like in the calcification experiments with or without periodic treatment with the PI3K/AKT/mTOR inhibitor Ly3023414 (Ly30). After 7 days, the cells were stained with Alizarin‐red. Shown are representative images of stained cells together with means ± SD from quantification of the red dye (n = 4, bars = 100 µm). B, In a parallel set up, calcium contents of the cultures were determined by the o‐cresolphtalein method (n = 4). C, Effects of Ly30 on the osteogenic transdifferentiation of VSMC were determined by qPCR after treatment of the cells as indicated. Shown are means ± SD (n = 4; *P <.05). D, VSMC were cultured like in the calcification experiments with or without periodic treatment with the MEK inhibitor UO126. After 7 days, the cells were stained with Alizarin‐red. Shown are representative images of stained cells (n = 3). In parallel, calcium contents of the cultures were determined by the o‐cresolphtalein method. Shown are means ± SD (n = 3; *P <.05)