Research Paper Volume 16, Issue 3 pp 2438—2456

Figure 4. Sp1 participated in β-GP-induced apoptotic protein levels and calcification via promoting Mark4 transcriptional activity in VSMCs. VSMCs were infected with pCDNA3.1-Sp1 or its control pCDNA3.1-GFP and lentiviral shRNA Mark4 (sh-Mark4) or its negative control (sh-NC) under normal medium. (A) Three transcription factors, including Sp1, directly targeting the Mark4 were screened using the online bioinformatics databases Animal TFDB and hTF target, as well as JASPAR. (B) RT–qPCR analysis of Mark4 expression in pCDNA3.1-Sp1 transfected VSMCs. (C) Western blot and quantitative densitometry analysis of Sp1 and Mark4 in VSMCs with pCDNA3.1-Sp1. ^*Means p < 0.05, ^**p < 0.01, ^***p < 0.001 vs. control. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 vs pCDNA3.1-GFP. (D) The putative Sp1 binding site and sequences in Mark4 promoter were shown. (E) ChIP assay verified that Sp1 was enriched at the Mark4 promoter region. (F, G) Western blot and quantitative densitometry analysis of Sp1, Mark4 and apoptosis-related proteins in VSMCs with pCDNA3.1-Sp1 and sh-Mark4. (H) Apoptosis of VSMCs with pCDNA3.1-Sp1 and sh-Mark4 was assessed by TUNEL staining. (I) Western blot and quantitative densitometry analysis of Runx2, (J) Alizarin Red S staining and (K) calcium content assessed calcification in VSMCs with pCDNA3.1-Sp1 and sh-Mark4. ^*Means p < 0.05, ^**p < 0.01, ^***p < 0.001 vs sh-NC, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 vs. pCDNA3.1-Sp1. Error bar represents three independent experiments.