Journal: The Journal of Clinical Investigation
Article Title: Epigenetic alteration of smooth muscle cells regulates endothelin-dependent blood pressure and hypertensive arterial remodeling
doi: 10.1172/JCI186146
Figure Lengend Snippet: ( A ) Schematic of the human JMJD3 gene, including gene structure, H3K27Ac, DHS, BP-associated SNPs, cloned fragments for luciferase assays, and vertebrate conservation (UCSC genome browser). ( B ) Luciferase results of DHS1 rs62059712 (T-major vs. C-minor) and ( C ) DHS2 rs74480102 (G-major vs. A-minor) in cultured primary human bronchial smooth muscle cells (HuBrSMCs). ( D ) qPCR for JMJD3 in HuSMCs with CRISPR/Cas9-mediated deletion of a 450 bp region encompassing rs62059712 within DHS1 (ΔDHS1) compared with unedited (WT) HuSMCs. ( E ) Sequence of rs62059712 T-major and C-minor sequences with SP1 consensus binding site underlined. ( F ) Western blot for SP1 after affinity purification using T versus C probes corresponding to rs62059712 SNP region incubated with human aortic smooth muscle cell (HuAoSMC) nuclear lysate. ( G ) ChIP-qPCR for Sp1 at the Jmjd3 promoter in mAoSMCs compared with IgG negative control. ( H ) ChIP-qPCR for Sp1 at the Jmjd3 promoter in mAoSMCs serum starved or treated with TGF-β. ( I ) Luciferase assays in mAoSMCs treated with siNTC and siSp1 siRNAs, then transfected with pGL3-DHS1-T and -C. ( J ) Luciferase assays in HuSMCs transfected with DHS1-C construct and then treated with TGF-β (20 ng/ml). ( K ) qPCR for Jmjd3 after NTC versus Sp1 knockdown in mAoSMCs serum starved for 16 hours. Data are represented as mean ± SEM. Results are representative of data from SMCs from 4–6 mice per group. n = 3 independent experiments. Two-tailed Student’s t test, * P < 0.05; ** P < 0.01.
Article Snippet: DNA fragments were then cloned into pGL3 luciferase basic vector (Addgene), and the correct sequence was verified by Sanger sequencing.
Techniques: Clone Assay, Luciferase, Cell Culture, CRISPR, Sequencing, Binding Assay, Western Blot, Affinity Purification, Incubation, ChIP-qPCR, Negative Control, Transfection, Construct, Knockdown, Two Tailed Test
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