Journal: Circulation

Article Title: Loss of Cardio-Protective Effects at the ADAMTS7 Locus Due to Gene-Smoking Interactions

doi: 10.1161/CIRCULATIONAHA.116.022069

Figure Lengend Snippet: (a) ADAMTS7 and CHRNB4-A3-A5 mRNA levels were measured in HCASMC. Cells were cultured to confluence, total RNA was extracted and cDNA generated. q-PCR was performed for ACTB, GAPDH, TBP, ADAMTS7, CHRNB4, CHRNA3, CHRNA5 (95°C 15s, 60°C 1min). Delta Cts were calculated as follows: (CtACTB + CtGAPDH + CtTBP)/3 – CtTARGET GENE). Fold changes are derived from delta delta Cts based on formula FC = 2−dCt. (b) Confluent HCASMC were exposed to cigarette smoke extract. Serum starved (x24 hrs.) confluent HCASMC were treated with 0.5% or 1.0% cigarette smoke extract (v/v) for 4, 12, and 24 hrs. in serum reduced conditions (0.5% FBS in DMEM). Total RNA was extracted, cDNA generated preparation and q-PCR performed for ADAMTS7 by Taqman and normalized to GAPDH. The Average Ct for ADAMTS7 at baseline was 28.25. Results were presented as means ± SEM, and data were analyzed using Student’s t-Test. (c) expression and eQTL Data from the GTEx consortium, the HapMap consortium (restricted to European populations), the Multiple Tissue Human Expression Resource (MuTHER) and in 147 donor HAoEC lines. Association of the independent lead variants identified in our conditional analyses with expression of ADAMTS7 and genes in the CHRNB4-A3-A5 cluster. A P-value threshold of 0.002 was set to account for multiple testing involved in the eQTL analyses.

Article Snippet: ChIP-seq experiments were performed on confluent HCASMC (Cell Applications 350-05a & Lonza CC-2583; cultured in SmGM-2 BulletKit media; Lonza) as described.

Techniques: Cell Culture, Generated, Derivative Assay, Expressing

Journal: Circulation

Article Title: Loss of Cardio-Protective Effects at the ADAMTS7 Locus Due to Gene-Smoking Interactions

doi: 10.1161/CIRCULATIONAHA.116.022069

Figure Lengend Snippet: Genome browser view of regulatory features at rs7178051 on Chr15q21.1. ChIP-seq experiments were performed on confluent HCASMC for TCF21, Jun, JunD, CEBP and H3K4me1, H3K27me3, H3K27ac. DNAaseI hypersensitivity data for human AoSMC were acquired from the ENCODE project. Human aortic tissue H3K4me1, H3K9me3, H3K27me3, and H3K36me3 ChIP-seq data were acquired from the NIH Roadmap Epigenomics Project. HCASMC = human coronary artery smooth muscle cells; AoSMC = human aortic smooth muscle cells.

Article Snippet: ChIP-seq experiments were performed on confluent HCASMC (Cell Applications 350-05a & Lonza CC-2583; cultured in SmGM-2 BulletKit media; Lonza) as described.

Techniques: ChIP-sequencing

Journal: Circulation

Article Title: Loss of Cardio-Protective Effects at the ADAMTS7 Locus Due to Gene-Smoking Interactions

doi: 10.1161/CIRCULATIONAHA.116.022069

Figure Lengend Snippet: (a) ADAMTS7 and CHRNB4-A3-A5 mRNA levels were measured in HCASMC. Cells were cultured to confluence, total RNA was extracted and cDNA generated. q-PCR was performed for ACTB, GAPDH, TBP, ADAMTS7, CHRNB4, CHRNA3, CHRNA5 (95°C 15s, 60°C 1min). Delta Cts were calculated as follows: (CtACTB + CtGAPDH + CtTBP)/3 – CtTARGET GENE). Fold changes are derived from delta delta Cts based on formula FC = 2−dCt. (b) Confluent HCASMC were exposed to cigarette smoke extract. Serum starved (x24 hrs.) confluent HCASMC were treated with 0.5% or 1.0% cigarette smoke extract (v/v) for 4, 12, and 24 hrs. in serum reduced conditions (0.5% FBS in DMEM). Total RNA was extracted, cDNA generated preparation and q-PCR performed for ADAMTS7 by Taqman and normalized to GAPDH. The Average Ct for ADAMTS7 at baseline was 28.25. Results were presented as means ± SEM, and data were analyzed using Student’s t-Test. (c) expression and eQTL Data from the GTEx consortium, the HapMap consortium (restricted to European populations), the Multiple Tissue Human Expression Resource (MuTHER) and in 147 donor HAoEC lines. Association of the independent lead variants identified in our conditional analyses with expression of ADAMTS7 and genes in the CHRNB4-A3-A5 cluster. A P-value threshold of 0.002 was set to account for multiple testing involved in the eQTL analyses.

Article Snippet: ChIP-seq experiments were performed on confluent HCASMC (Cell Applications 350-05a & Lonza CC-2583; cultured in SmGM-2 BulletKit media; Lonza) as described.

Techniques: Cell Culture, Generated, Derivative Assay, Expressing