Figure 1.

BCL11B (B-cell leukemia 11b) is expressed in vascular smooth muscle (VSM). A, A conserved region within the 3′-BCL11B genetic locus with SNPs highly associated with arterial stiffness is present in murine aortas and identified as AI060616 (National Center for Biotechnology Information [NCBI]). AI060616 356 bp nucleotide sequence was confirmed by sequencing polymerase chain reaction (PCR) products from aortic DNA obtained from 2 C57Bl/6J mice (1 and 2). Sequencing chromatogram tracing shown in lower panel. B, Representative fluorescent images of aortic sections, taken at 20× and 40× magnification, indicating Bcl11b’s localization in VSM. Red fluorescence indicates tomato (mT) expression in lieu of Bcl11b, upon tamoxifen-induced Bcl11b removal and mT induction in 3 ER-Cre-Bcl11b^flox/flox-mTomato mice. Asterisks indicate clusters of VSM cells with high mT fluorescence intensity. C, Representative images (40× magnification) of human primary aortic smooth muscle cell immunostaining with anti-BCL11B; rabbit IgG serves as negative control for antibody specificity. DAPI indicates nuclei. n=3 replicates with 3 different cell lines, as described in materials and methods. D, Western blot on murine aortas confirmed Bcl11b protein expression, with a band of expected MW 100–120 kDa. β-actin serves as loading control. Each lane represents one mouse. E, Image of 1% agarose gel electrophoresis of qRT-PCR Taqman products indicates BCL11B mRNA expression in human aortas (n=9). Each lane represents one human subject. β-ACTIN used as endogenous housekeeping gene in multiplexed assay. F, Relative luciferase activity (RLA), expressed as a ratio with Renilla luciferase, in VSM cells transfected with luciferase reporter plasmids expressing an empty plasmid (pGL3) or Bcl11b promoter (pGL3-pr3). Results represent 3 replicate experiments. *P=2.0×10⁻² by unpaired t test.