Fig. 3: Knockdown of NEXN aggravates vascular calcification in vitro and in vivo.

a–d NEXN siRNA pre-transfected human VSMCs treated with GM or CM for 7 days (n = 4 biological replicates). a Alizarin red staining of human VSMCs. Scale bar: 500 μm. b, c Western blot and quantification of the protein expression of osteogenic markers Collagen I, RUNX2 and BMP2 in human VSMCs (n = 4 biological replicates; error bar: mean (centre) ± standard deviation; one-way ANOVA and Turkey’s post hoc test were used for comparisons among more than two groups). d Immunofluorescence for NEXN (green) and RUNX2 (red) in human VSMCs. Scale bar: 25 μm. e–l Nexn^F/F and Nexn^ismKO mice (8-week-old male) were fed with AP or normal diet for 16 weeks (n = 6 biological replicates). e Schematic diagram of generating Nexn^F/F; Myh11^Cre/ERT2 (Nexn^ismKO) male mice. f Schematic diagram of vascular calcification induced by AP-diet in mice (8-week-old male). g Whole-body calcified vessels in mice were scanned by micro-CT and reconstructed in three dimensions. Scale bar: 5 mm. h Quantification of the calcification surface of the mice vessels (n = 6 biological replicates; error bar: mean (centre) ± standard deviation; Mann-Whitney U test was used for comparisons). i Quantification of the calcification volume in mice vessels (n = 6 biological replicates; error bar: mean (centre) ± standard deviation; Mann-Whitney U test was used for comparisons). j Measurement of the pulse wave velocity (PWV) in the mice arteries (n = 6 biological replicates; error bar: mean (centre) ± standard deviation; one-way ANOVA and Turkey’s post hoc test were used for comparisons among more than two groups). k, l Western blot and quantification of the protein expression of osteogenic markers RUNX2 and BMP2 in mice aortas (n = 6 biological replicates; error bar: mean (centre) ± standard deviation; one-way ANOVA and Turkey’s post hoc test were used for comparisons among more than two groups). GM, growth medium. CM, calcification medium. The graphic (e) was created using Adobe Illustrator 2021. The graphic (f) was created using Adobe Illustrator 2021, with icons from Servier Medical Art, which is licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/). Source data are provided as a Source Data file.