Journal: Nature Communications

Article Title: Integrative functional genomics identifies regulatory mechanisms at coronary artery disease loci

doi: 10.1038/ncomms12092

Figure Lengend Snippet: ( a ) ATAC-seq signal-to-noise ratio, calculated as distribution of ATAC-seq reads centred on TSS in a 1,000-bp window, normalized to total mapped reads and comparing 50 and 100 million mapped reads from representative data sets ( n =22 biological replicates). ( b ) Scatter plot showing correlation of ATAC-seq tag intensities from two independent biological replicates ( r 2 ∼0.94). ( c ) Histogram distribution and resulting P value (via cumulative binomal distribution) for top four enriched motifs identified using de novo motif enrichment analysis in open chromatin peaks in HCASMCs treated with TGF-β1 ( n =2 biological replicates per condition). ( d ) Similar results shown above for HCASMCs treated with PDGF-BB. ( e ) Hierarchical clustering of open chromatin normalized read counts in stimulated HCASMCs from two independent donors (1 and 2), with clustering on CAD loci annotated to transcription start site of nearest gene. ( f ) Genomic Regions Enrichment of Annotations Tool (GREAT) analysis of stimulated HCASMC open chromatin regions overlapping entire GWAS catalogue ( n =2 biological replicates), showing enrichment for Disease Ontologies relative to whole-genome background. P values were calculated using a combination of binomial and hypergeometric tests.

Article Snippet: Primary HCASMCs derived from normal human donor hearts were purchased from three different manufacturers, Lonza, PromoCell and Cell Applications (all tested negative for mycoplasma contamination).

Techniques:

Journal: Nature Communications

Article Title: Integrative functional genomics identifies regulatory mechanisms at coronary artery disease loci

doi: 10.1038/ncomms12092

Figure Lengend Snippet: ( a ) Venn diagram of overlapping 5240 candidate CAD-associated variants (including those in high linkage disequilibrium at r 2 ≥0.8) with HCASMC ATAC-seq open chromatin regions ( n =323), H3K27ac ChIP-seq active enhancer regions ( n =462) or TF binding via TCF21 or AP-1 ChIP-seq ( n =193). Unique overlapping numbers shown for combined overlaps, respectively. ( b ) Forest plot depicting odds ratio (OR) of enrichment for CARDIoGRAMplusC4D (CAD), inflammatory bowel disease (IBD), ulcerative colitis (UC) or entire GWAS catalogue SNPs in individual or combined HCASMC data sets as calculated using the Fisher's exact test. Dots represent mean OR and lateral lines represent 95% confidence intervals. ( c ) Histogram distribution of globally normalized GWAS SNPs in regions centred on HCASMC open chromatin regions within a 1-kb window. ( d ) Heatmap distribution of HCASMC open chromatin regions centred on CTCF motif (from JASPAR) within a 0.5-kb window (left panel). Hierarchical clustering heatmap showing distribution of ATAC-seq open chromatin, JUN or TCF21 ChIP-seq binding regions centred on 5,240 CARDIoGRAMplusC4D SNPs (right panels). ( e ) Two-dimensional scatter plot of GWAS SNPs in HCASMC open chromatin regions showing most significant enrichment for cardiovascular (CARDIoGRAMplusC4D and coronary heart), brain and autoimmune phenotypes in upper right quadrant. Data shown are representative of n =10 biological replicates in HCASMCs cultured under normal growth conditions.

Article Snippet: Primary HCASMCs derived from normal human donor hearts were purchased from three different manufacturers, Lonza, PromoCell and Cell Applications (all tested negative for mycoplasma contamination).

Techniques: ChIP-sequencing, Binding Assay, Cell Culture

Journal: Nature Communications

Article Title: Integrative functional genomics identifies regulatory mechanisms at coronary artery disease loci

doi: 10.1038/ncomms12092

Figure Lengend Snippet: ( a ) Scatter plot showing correlation of ATAC-seq tag intensities from normal coronary artery tissue and quiescent HCASMCs in serum-free conditions ( r 2 =0.70). Similar results were observed from n =3 biological replicates. ( b ) Principal component analysis of ATAC-seq open chromatin peaks from normal and atherosclerotic coronary artery tissues and HCASMCs cultured under various conditions. Principal component 1 was excluded as it depicted a batch effect between experiments. Principal component 2 (PC2; x axis) represents the effect of the cell line used for treatment with various factors. Principal component 3 (PC3; y axis) represents the effect of the treatment with various factors and also partially separates normal and athero tissues (denoted by dashed line). ( c ) Histogram showing de novo motif distribution in ex vivo open chromatin peaks and resulting enrichment analysis in ex vivo open chromatin regions from atherosclerotic coronary artery tissues. Data represent n =2 biological replicates. ( d – e ) Histogram density plot showing CAD lead and LD SNPs enriched in matrices for the classic AP-1 motif, TGANTCA, versus randomized AP-1 matrices (AP-1 control). Randomized CAD-matched SNPs centred on AP-1 or AP-1-randomized control matrices. Count densities were globally normalized by the total number of counts for both SNP and AP-1 motif regions in the 1-kb window.

Article Snippet: Primary HCASMCs derived from normal human donor hearts were purchased from three different manufacturers, Lonza, PromoCell and Cell Applications (all tested negative for mycoplasma contamination).

Techniques: Cell Culture, Ex Vivo, Control

Journal: Nature Communications

Article Title: Integrative functional genomics identifies regulatory mechanisms at coronary artery disease loci

doi: 10.1038/ncomms12092

Figure Lengend Snippet: ( a ) LocusZoom plot showing results of CARDIoGRAMplusC4D 1000G fine-mapping at SMAD3 locus at chromosome 15q24.1 ( n =60,801 cases; n =123,504 controls) . Circles represent SNPs associated using an additive or recessive model, and colour-coded for LD ( r 2 ) with the lead SNP, rs56062135 (purple diamond), based on 1000G phase 1 v3 training data set. ( b ) LD plot generated from 1000G phase 1 augmented haplotypes in Europeans for SMAD3 locus, showing linked lead SNP, rs56062135, with candidate regulatory SNP, rs17293632. Colour-coded for LD based on D ′ values, shown as in boxes. ( c ) UCSC browser screenshot at SMAD3 locus, showing overlap of candidate SNP rs17293632 with ATAC-seq open chromatin tracks in coronary tissue ( n =3 biological replicates per condition) and HCASMCs treated under various conditions ( n =2 biological replicates per condition), TF-binding ChIP-seq tracks for TCF21, JUN and JUND, and active enhancer histone modification H3K27ac ChIP-seq ( n =4 biological replicates), as well as ENCODE layered H3K27ac for HUVEC (blue) and NHLF cells (purple). Inset, motifs in open chromatin regions showing alignment to reference sequence and position relative to rs17293632. Genomic coordinates refer to hg19 assembly. ( d ) Normalized ATAC-seq read counts for HCASMCs treated under various conditions and by genotype at rs17293632. Values represent mean±s.e.m. ( n =2 biological replicates for stimulations and n =5 biological replicates for different genotypes). ( e ) Allele-specific ChIP (haploChIP) for AP-1 proteins (JUN, JUNB and ATF3), TCF21 and H3K27ac in HCASMCs heterozygous at rs17293632. Values represent mean±s.e.m. of triplicates from a representative experiment ( n =5 biological replicates). * P <0.01 versus control, IgG or between two genotypes using an unpaired two-tailed t -test with Welch's correction for unequal variances.

Article Snippet: Primary HCASMCs derived from normal human donor hearts were purchased from three different manufacturers, Lonza, PromoCell and Cell Applications (all tested negative for mycoplasma contamination).

Techniques: Generated, Binding Assay, ChIP-sequencing, Modification, Sequencing, Control, Two Tailed Test

Journal: Nature Communications

Article Title: Integrative functional genomics identifies regulatory mechanisms at coronary artery disease loci

doi: 10.1038/ncomms12092

Figure Lengend Snippet: ( a ) UCSC browser screenshot at 9p21.3/ CDKN2B-AS locus, showing overlap of candidate SNP rs1537373 with ATAC-seq open chromatin tracks in coronary tissue ( n =3 biological replicates per condition) and HCASMCs treated under various conditions ( n =2 biological replicates per condition), TF-binding ChIP-seq tracks for TCF21, JUN and JUND, and active enhancer histone modification H3K27ac ChIP-seq ( n =4 biological replicates), as well as ENCODE layered H3K27ac for HUVEC (blue) and NHLF cells (purple). Inset, motifs generated from HOMER in open chromatin regions showing alignment to reference sequence and position relative to rs1537373 SNP. Genomic coordinates refer to hg19 assembly. ( b ) Normalized ATAC-seq read counts for HCASMCs treated under various conditions and by genotype at rs1537373. Values represent mean±s.e.m. ( n =2 biological replicates for stimulations and n =5 biological replicates for different genotypes). ( c ) Allele-specific ChIP (haploChIP) for AP-1 proteins (JUN, JUNB and ATF3), TCF21 and H3K27ac in HCASMCs heterozygous at rs1537373. Values represent mean±s.e.m. of triplicates from a representative experiment ( n =5 biological replicates). * P <0.01 versus control, IgG or between two genotypes using an unpaired two-tailed t -test with Welch's correction for unequal variances.

Article Snippet: Primary HCASMCs derived from normal human donor hearts were purchased from three different manufacturers, Lonza, PromoCell and Cell Applications (all tested negative for mycoplasma contamination).

Techniques: Binding Assay, ChIP-sequencing, Modification, Generated, Sequencing, Control, Two Tailed Test

Journal: Nature Communications

Article Title: Integrative functional genomics identifies regulatory mechanisms at coronary artery disease loci

doi: 10.1038/ncomms12092

Figure Lengend Snippet: ( a ) Results of enhancer trap assay for seven candidate SNPs cloned into minimal promoter-driven luciferase reporter vector pLuc-MCS, and transfected in A7r5 smooth muscle cell line. ( b ) Results of enhancer trap assay for rs17293632-C/T ( SMAD3 ) reporters (rs172_C-Luc and rs172_T-Luc) versus consensus AP-1-Luc (3 ×) reporter with co-transfection of expression constructs for JUN, JUNB or JUND in A7r5 SMC. ( c ) Results of enhancer trap assay for rs17293632 reporters with siRNA-mediated knockdown of JUN, JUNB or JUND in HCASMCs. ( a – c ). Values represent mean±s.e.m. of triplicates for a representative experiment ( n =4 biological replicates), expressed as fold change relative to pLuc-MCS. * P <0.01 between allele-specific reporters using a two-tailed Student's t -test. ( d ) SMAD3 gene expression levels in HCASMCs with respect to genotype at rs17293632, expressed as ΔΔCt values normalized to GAPDH levels (fold change). Values represent mean±s.e.m. of triplicates ( n =64 independent donors/biological replicates). P values calculated using a Welch's unequal variances t -test. ( e ) Allelic expression imbalance for candidate regulatory SNP rs17293632 at SMAD3 detected by TaqMan qPCR in HCASMC pre-mRNA from heterozygous individual donors ( n =23). Values represent mean±s.e.m. of triplicates for cDNA ratio normalized to gDNA ratio in heterozygous individuals at rs17293632. P values shown represent comparison of AEI from all samples versus expected allelic ratio of 1.0 using a Welch's unequal variances t -test. ** P <0.001, *** P <0.0001 for individual samples of allelic imbalance ratio versus expected allelic ratio of 1.0. ( f ) Scatter plot for the most significant differentially expressed genes from RNA-seq DEseq analysis of HCASMCs treated with serum free (control) or PDGF-BB for 1 h ( n =2 biological replicates per condition). –log10( P values) and log2(fold change) determined as described in the Methods section. Labels are shown for immediate early response genes.

Article Snippet: Primary HCASMCs derived from normal human donor hearts were purchased from three different manufacturers, Lonza, PromoCell and Cell Applications (all tested negative for mycoplasma contamination).

Techniques: TRAP Assay, Clone Assay, Luciferase, Plasmid Preparation, Transfection, Cotransfection, Expressing, Construct, Knockdown, Two Tailed Test, Gene Expression, Comparison, RNA Sequencing, Control

Journal: International Journal of General Medicine

Article Title: Phosphoglycerate Dehydrogenase Overexpression Inhibits Ferroptosis to Repress Calcification of Human Coronary Artery Vascular Smooth Muscle Cells via the P53/SLC7A11 Pathway

doi: 10.2147/IJGM.S473908

Figure Lengend Snippet: PHGDH overexpression inhibited the calcification level of calcifying medium (CM)-induced human coronary artery smooth muscle cells (HCASMCs). ( A ) T he relative mRNA and protein expression levels of PHGDH. ( B ) The cell viability. ( C ) The results of alizarin red staining. ( D ) The protein expression level of RUNX2 and bone morphogenetic protein 2 (BMP2). ( E ) The relative ALP activity. ( F ) The calcium content. **p < 0.01 vs the Normal group; ## p < 0.01 vs the oe-NC group.

Article Snippet: Primary HCASMCs were purchased from iCell Bioscience Inc. (Shanghai, China).

Techniques: Over Expression, Expressing, Staining, Activity Assay

Journal: International Journal of General Medicine

Article Title: Phosphoglycerate Dehydrogenase Overexpression Inhibits Ferroptosis to Repress Calcification of Human Coronary Artery Vascular Smooth Muscle Cells via the P53/SLC7A11 Pathway

doi: 10.2147/IJGM.S473908

Figure Lengend Snippet: Inhibition of ferroptosis suppressed the calcification level of CM-induced HCASMCs. ( A ) The results of alizarin red staining. Fer-1: ferrostatin-1. ( B ) The calcium content. ( C ) The protein expression level of RUNX2 and BMP2. ( D ) The expression level of glutathione (GSH). ( E ) The protein expression levels of cyclooxygenase 2 (COX2) and glutathione peroxidase 4 (GPX4). **p < 0.01 vs the Normal group; ## p < 0.01 vs the Control group.

Article Snippet: Primary HCASMCs were purchased from iCell Bioscience Inc. (Shanghai, China).

Techniques: Inhibition, Staining, Expressing, Control

Journal: International Journal of General Medicine

Article Title: Phosphoglycerate Dehydrogenase Overexpression Inhibits Ferroptosis to Repress Calcification of Human Coronary Artery Vascular Smooth Muscle Cells via the P53/SLC7A11 Pathway

doi: 10.2147/IJGM.S473908

Figure Lengend Snippet: PHGDH overexpression inhibited the calcification level of CM-induced HCASMCs by inhibiting ferroptosis. ( A ) The results of alizarin red staining. ( B ) The calcium content. ( C ) The relative ALP activity. ( D ) The expression level of GSH. (E) The protein expression level of COX2 and GPX4. **p < 0.01.

Article Snippet: Primary HCASMCs were purchased from iCell Bioscience Inc. (Shanghai, China).

Techniques: Over Expression, Staining, Activity Assay, Expressing