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. 2018 Jan 4;7(1):e007405. doi: 10.1161/JAHA.117.007405

Figure 7.

Figure 7

IKKβ regulates vascular smooth muscle cells (VSMC) calcification through its kinase‐independent function. A, Representative Western blots and densitometric analysis of active β‐catenin/GAPDH and Runt‐related transcription factor 2 (Runx2)/GAPDH expression in cultured VSMCs isolated from wild‐type (WT) mouse with stimulation by interleukin (IL)‐1β (2.5 ng/mL) or the vehicle for 15 minutes. Bars represent the mean±SD (t test, n=3). B, Kinase‐dead IKKβ transgene construct. C, Representative Western blots and densitometric analysis of IKKβ/GAPDH, phosphorylated p65/total p65 expression in VSMCs isolated from kinase dead (KD) and IKKβ knockout (KO) mice. Bars represent the mean±SD (t test, n=3). D, Representative microscopy images of Alizarin Red staining of 4‐week cultured VSMCs isolated from WT and KD mice and quantification of VSMC calcification. Calcification was quantified by ImageJ software. Graph presented is the percentage of positively stained area in the total area randomly selected. Bars represent the mean±SD (t test, n=6). E, Results of quantitative real‐time PCR (qRT‐PCR) for the expression of various osteogenic‐related genes (osterix, alkaline phosphatase [ALP], and osteocalcin) in WT, KD, KO, and kinase active IKKβ (KA) cells that were normalized to the Rn18s mRNA level. WT samples used in RT‐PCR were from littermate of KO mouse. Cells used in qRT‐PCR were cultured for 2 weeks in normal medium with 10% fetal bovine serum. Bars represent the mean±SD (1‐way ANOVA, n=4). **P<0.01, ***P<0.001.