Fig. 2. Silencing of ENPP1 by siRNA in hiPSC-derived VSMCs results in a reduction of ENPP1 RNA expression and an increase in hiPSC-derived VSMCs proliferation.

SMC markers calponin (first panel) and SM-MHC11 (second panel) confirmed the SMC phenotype of hiPSC-derived VSMCs (a). hiPSC-derived VSMCs were transfected overnight with ENPP1 siRNA or control siRNA, followed by 24-h starvation in 0.25% FBS. Then, the cells were cultured in basal media. ENPP1 RNA expression (b) and hiPSC-derived VSMCs cell number (c), and proliferation (d) were investigated. b, c Values are presented as the mean ± SEM, n = 4; ^***p < 0.001 vs. control nontargeting siRNA transfected cells. d Cell proliferation was evaluated by [³H] thymidine uptake. [³H]thymidine was added in the last 18 h of culture. The results are expressed as the CPM ± SEM, n = 4. ^***p < 0.001 vs. control nontargeting siRNA transfected cells (Student’s t test, unpaired two-group testing for means)