Fig. 3.

Effects of bisphosphonates (a), AMP (b), adenosine (c), and rhENPP1-Fc (d) on accelerated proliferation in ENPP1-silenced hiPSC-derived VSMCs. hiPSC-derived VSMCs were transfected overnight with ENPP1 siRNA or control siRNA, followed by 24-h starvation in 0.25% FBS. Then, cells were cultured in basal media, supplemented with etidronate (0.1–100 µM, a), AMP (0.3–300 µM, b), and adenosine (0.3–300 µM, c) for 3 days before determining proliferation. Cell proliferation was evaluated by [³H] thymidine uptake. [³H]thymidine was added in the last 18 h of culture. Extracellular PP_i levels in the cell culture medium of cells supplemented with 300 µM ATP and rhENPP1-Fc (0.01–5 µg/ml, d) were determined (e). Values are presented as the mean ± SEM, n = 4. ^***p < 0.001 (one-way ANOVA multiple group comparison followed by the Bonferroni’s post hoc test)