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. 2020 Jul 8;34(8):11087–11100. doi: 10.1096/fj.202001062R

FIGURE 5.

FIGURE 5

Drugs identified by network analysis ameliorate vascular smooth muscle cell calcification. A, Human coronary artery smooth muscle cells (HCSMCs) were treated with everolimus, pomalidomide, or temsirolimus for 10 days. Calcification was assessed visually using Alizarin red staining (top) and semiquantitatively (bottom) (n = 5). B, Western immunoblotting of HCSMCs exposed to calcification medium and drugs for 24 hours demonstrated that the drugs interacted with their respective network targets. Since everolimus and temsirolimus inhibit mTOR activity, P70S6K activation was also measured as an indicator of effective inhibition (n = 5‐6). C, Western immunoblotting of HCSMCs exposed to calcification medium and drugs for 7 days demonstrated a decrease in the expression of the osteoblast transcription factor Runx2 (top) (n = 3) and a decrease in alkaline phosphatase activity (bottom) (n = 5). D, Cell proliferation (top) and apoptosis (bottom) were examined in HCSMC exposed to calcification medium and drugs for 10 days to show that observed differences in calcification did not the result from changes in cell number or viability. CTL, control; CM, calcification medium; Ev, everolimus (20 nmol/L); Pom, pomalidomide (10 µmol/L); Tem, temsirolimus (10 nmol/L).