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. 2023 Aug 31;43(10):1967–1989. doi: 10.1161/ATVBAHA.123.319925

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© 2023 The Authors.

Arteriosclerosis, Thrombosis, and Vascular Biology is published on behalf of the American Heart Association, Inc., by Wolters Kluwer Health, Inc. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial-NoDerivs License, which permits use, distribution, and reproduction in any medium, provided that the original work is properly cited, the use is noncommercial, and no modifications or adaptations are made.

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Figure 4. — Identification of the dynamically bound target RNAs of hnRNP H1 (heterogeneous nuclear ribonucleoprotein H1) using RNA immunoprecipitation sequencing (RIP-seq) and RNA-sequencing (RNA-seq). A, Schematic overview of RIP-seq design. Libraries were generated from input and hnRNP H1 immunoprecipitation (IP) samples±TGF (transforming growth factor)-β stimulation to identify global and TGF-β–regulated binding patterns of hnRNP H1. B, Target RNAs of hnRNP H1. Proportion peak-associated genes identified as targets of hnRNP H1, sorted by gene type. C, Proportion of TGF-β–regulated binding. Proportion of hnRNP H1 target genes differentially bound after TGF-β stimulation (P<0.05). D, Pathway enrichment of hnRNP H1 targets. Enriched terms (KEGG pathway) among all peak-related genes (all targets) and differentially bound peak-related genes (TGF-β–regulated binding). E, mRNA summit location. The proportion of summit locations identified within mRNA targets±TGF-β stimulation (from averaged replicates). F, Predicted binding motifs of hnRNP H1. Significantly enriched motifs were identified in peaks in the presence or absence of TGF-β stimulation. G, Heatmap of effects of hnRNP H1 knockdown on RNA expression. RNA-seq after siRNA knockdown (48 hours) of hnRNP H1 in the absence (0 ng/mL TGF-β, siCtl_NS vs sihnRNP_NS) or presence (10 ng/mL TGF-β, siCtl_TGF vs sihnRNP_TGF) of TGF-β stimulation. H, Pathway enrichment of differentially expressed genes si-Ctl vs si-hnRNP H1 in nonstimulated cells. Pathway enrichment among all differentially expressed genes, upregulated and downregulated genes in si-hnRNP H1 samples compared with si-Ctl samples in the absence of TGF-β stimulation. I, Pathway enrichment of differentially expressed genes si-Ctl vs si-hnRNP H1 in TGF-β stimulated cells. Pathway enrichment among all differentially expressed genes, upregulated and downregulated genes in si-hnRNP H1 samples compared with si-Ctl samples in the presence of TGF-β stimulation. K, Overlap between RIP-seq and RNA-seq. The overlap between peak-associated genes identified as targets of hnRNP H1 (RIP-seq) compared with differentially expressed genes (si-Ctl- vs si-hnRNP H1, P<0.05 RNA-seq) and genes with significant alternative splicing (si-Ctl- vs si-hnRNP H1, P<0.05 RNA-seq). AGE-RAGE indicates advanced glycation end-products-receptor for AGE; CDS, coding sequence; DEG, differentially expressed gene; ECM, extracellular matrix; HIF, hypoxia inducible factor; KEGG, Kyoto Encyclopedia of Genes and Genomes; lncRNA, long noncoding RNA; MAPK, mitogen-activated protein kinase; MCEC, mouse cardiac endothelial cell; metab., metabolism; mTOR, mammalian target of rapamycin; siCtl_NS and siCtl_TGF, control siRNA nonstimuated and control siRNA TGF-β stimulated; sihnRNP_NS and sihnRNP_TGF, hnRNP H1 siRNA nonstimulated and hnRNP H1 siRNA TGF-β stimulated; siRNA, small interfering RNA; tRNA, transfer RNA; and UTR, untranslated region.