Fig. 3.

A-EC/sEVs disrupted the smad1/5/9 signalling pathway by delivering miR-126-5p. A Heatmap showing expression profiles of candidate miRNAs. B Analysis by PCR showing normalized levels of candidate miRNAs. C The sEVs were treated or not (Con) with Triton X-100 (T) and incubated with or without RNases and PK (PK/R). miR-126 levels were measured by qPCR. D Venn diagram showing bioinformatics analysis of miR-126-5p target genes. E Luciferase reporter assays were conducted using luciferase constructs carrying a WT or mutant BMPR1B 3’-UTR co-transfected into HA-VSMCs with miR-126-5p mimics. Firefly luciferase activity was normalised to Renilla luciferase activity. Data were presented as mean ± SD of three replicates. ^##P < 0.01. F Expression of BMPR1B, Runx2, BMP2, t-p-smad1/5/9 and p-smad1/5/9 after transfection with miR-126-5p in calcified HA-VSMCs. G, H The representative image of ALP staining and ALP activity. G, I The representative image of ARS staining and calcium deposition. J–K Silencing efficiency of small interfering RNA on BMPR1B analyzed by qPCR and western blotting. L The protein level of t-p-smad1/5/9, intranuclear p-smad1/5/9, Runx2 and BMP2 was detected by western blotting after miR-126-5p transfection in a BMPR1B-silenced VSMC calcification model. M The representative image of ALP staining and ARS staining after miR-126-5p transfection in a BMPR1B-silenced VSMC calcification model. ALP activity (N) and calcium deposition (O) after miR-126-5p transfection in a BMPR1B-silenced VSMC calcification model. Data were presented as mean ± SD of three replicates. NS, not significant