Fig. 2: NEXN expression was downregulated during vascular calcification in vitro and in vivo.

a Alizarin red staining of human VSMCs cultured for 7 days in GM or CM. Scale bar: 500 μm. b Quantitative analysis of alizarin red by microplate reader obtained from (a) (n = 4 biological replicates; error bar: mean (centre) ± standard deviation; statistical significance was assessed using two-sided and unpaired t-test). c Quantitative analysis of calcium content in human VSMCs cultured for 7 days in GM or CM (n = 4 biological replicates; error bar: mean (centre) ± standard deviation; statistical significance was assessed using two-sided and unpaired t-test). d Representative immunofluorescence for NEXN (green) and RUNX2 (red) in human VSMCs cultured for 7 days in GM or CM. Scale bar: 25 μm. e, f Western blot and quantification of the protein expression of NEXN, osteogenic markers Collagen I, RUNX2 and BMP2 in human VSMCs cultured for 7 days in GM or CM (n = 4 biological replicates; error bar: mean (centre) ± standard deviation; statistical significance was assessed using two-sided and unpaired t-test). g Alizarin red staining of the entire aortas of male C57BL/6 mice subjected to VitD3-induced vascular calcification. Scale bar: 5 mm. h Quantification of the positive area of alizarin red staining in the entire aortas obtained from (g) (n = 6 biological replicates; error bar: mean (centre) ± standard deviation; statistical significance was assessed using Mann-Whitney U test). i Quantitative analysis of calcium content in the entire aortas of male C57BL/6 mice subjected to VitD3-induced vascular calcification (n = 6 biological replicates; error bar: mean (centre) ± standard deviation; statistical significance was assessed using two-sided and unpaired t-test). j, k Western blot and quantification of the protein expression of NEXN and the osteogenic marker BMP2 in the aortas of male C57BL/6 mice subjected to VitD3-induced vascular calcification (n = 6 biological replicates; error bar: mean (centre) ± standard deviation; statistical significance was assessed using two-sided and unpaired t-test). l, m The compliance of the aortas was measured via echocardiography in the male C57BL/6 mice with a 5/6 nephrectomy-induced vascular calcification (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). n, o Western blot and quantification of the protein expression of NEXN, osteogenic markers RUNX2 and BMP2 in aortas from the male C57BL/6 mice with a 5/6 nephrectomy-induced vascular calcification (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). D diastolic diameter, S systolic diameter, GM growth medium, CM calcification medium. Source data are provided as a Source Data file.