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. 2024 Oct 17;23:369. doi: 10.1186/s12933-024-02440-7

Fig. 4.

Fig. 4

hsa_circ_0008362 interacted directly with Runx2 to promote VSMC calcification. A hsa_circ_0008362 had strong binding region with Runx2 protein. B The expression of Runx2 was measured by WB after biotin‐labelled circ_0008362 pull‐down assay. C, D The expression of hsa_circ_0008362 and mFBXW4 detected by qRT-PCR in VSMCs via anti-Runx2 RIP experiments, respectively. E The half-life of Runx2 protein was measured by WB in VSMCs treated with ECNG-EVs or ECHG-EVs, accompanied with CHX (5ug/ml) treatment. F The degradation of Runx2 protein was measured by WB in VSMCs treated with hsa_circ_0008362 plasmid, accompanied with CHX (5 ug/ml) treatment. G The level of Runx2 protein was measured by WB in VSMCs with different treatment. H ARS staining showed the mineralized nodules in VSMCs with different treatment, and the calcium content was quantified by spectrophotometry. One-way ANOVA with Tukey’s multiple comparisons test (B, C, D, H, G) was used. one-way ANOVA and Student’s t-test (E, F) were used. Three independent experiments were performed, and representative data were shown. Data were shown as mean ± SD. ****p < 0.0001, ***p < 0.001, **p < 0.005. ns, no significance; WB, western blot; ECNG-EVs, EVs derived from normal-glucose induced ECs; ECHG-EVs, EVs derived from high-glucose induced ECs; circ_0008362-WT, circ_0008362-wild type plasmid; ARS, alizarin red s; CHX, cycloheximide