Journal: Nature communications

Article Title: Stress-induced RNA-chromatin interactions promote endothelial dysfunction.

doi: 10.1038/s41467-020-18957-w

Figure Lengend Snippet: Fig. 1 High glucose and TNFα induce a profound gene expression and phenotypic change. a HUVECs were treated in biological duplicates as Day 0: 25 mM mannitol as normal glucose and osmolarity control (NM); Day 3: combined treatment consisting 25 mM D-glucose and 5 ng/mL TNFα (H + T) for 3 days; and Day 7: H + T treatment for 7 days. Each group of treated cells was subjected to single-cell RNA-seq (scRNA-seq), Hi-C, and iMARGI assays. b t-SNE plot of scRNA-seq (4000–15,000 cells per sample) showed clear separation by treatment condition into three distinct clusters. c Principal component analysis of scRNA-seq data: single cells are plotted in the first two PC space and are labeled in red (Day 0, i.e., NM), green (Day 3, i.e., 3-day H + T treatment), and blue (Day 7, i.e., 7-day H + T treatment). d Expression heatmap (z-scaled) of top DE genes in single ECs grouped into functional pathways. Cells were ordered by increasing SERPINE1 expression (per each sample separately) and binned per 100 cells for the analysis. A total of 269 bins in Day 0, 177 bins in Day 3, and 148 bins in Day 7. e t-SNE plots of the expression level of selected genes in each single cell across the time course. The RNA levels are represented by log-normalized unique molecular identifier counts. f mRNA levels of eNOS and α-SMA in NM vs. H + T-treated HUVECs and cells untreated (NT) or treated with TGF-β (10 ng/mL) and IL-1β (5 ng/mL; T + I) for 3 or 7 days. The respective control was set as 1. Relative eNOS level: data represent mean ± SEM from five independent experiments; relative α-SMA level in H + T treatment: data represent mean ± SEM from seven independent experiments; relative α-SMA level in T + I treatment: data represent mean ± SEM from four independent experiments. *P = 0.0067, 0.0087, 0.0057, and 0.0017 from left to right based on ANOVA with Bonferroni as post hoc test. g Cell morphology under bright field (BF), immunofluorescent staining of α-SMA, and VE-cadherin (VE-cad), phalloidin staining of cytoskeleton, and DRAQ5 (DRAQ) staining of the nuclei. Representative images from five independent experiments are shown. Scale bar of BF = 100 µm; scale bars of (immuno)fluorescent staining = 50 µm. Source data are provided as a Source data file.

Article Snippet: Plasmid transfection was performed using the CytofectTM HUVEC Transfection kit (Cell Applications) following the manufacturer’s protocol in 6-well or 12-well plates.

Techniques: Gene Expression, Control, RNA Sequencing, Hi-C, Labeling, Expressing, Functional Assay, Staining

Journal: Nature communications

Article Title: Stress-induced RNA-chromatin interactions promote endothelial dysfunction.

doi: 10.1038/s41467-020-18957-w

Figure Lengend Snippet: Fig. 2 Overview of time-course Hi-C and iMARGI data. a, b Proportions of intrachromosomal (yellow) and interchromosomal read pairs (blue) in Hi-C (a) and iMARGI data (b) at the three time points (columns). c An example of interchromosomal iMARGI read pairs mapped to chromosome 2 near LINC00607 (left) and chromosome 7 near SERPINE1 (right). The RNA end (pink) and the DNA end (green) of each read pair is linked by a horizontal line. d Examples of overlapping iMARGI read pairs on a contact matrix from the RNA end (rows) to the DNA end (columns) with SEs (marked in light blue and in SE tracks) in control (Day 0) ECs. Genome region: chr1:75,000,0000–chr1:125,000,000. Resolution = 200 kb. e Proportions of iMARGI read pairs with the RNA ends (pink) or the DNA ends (green) in Day 0 (Ctrl) and Days 3 and 7 (H + T) ECs mapped to HUVEC SEs. Dotted line: the relative size of HUVEC SEs compared to the size of the genome. Source data are provided as a Source data file.

Article Snippet: Plasmid transfection was performed using the CytofectTM HUVEC Transfection kit (Cell Applications) following the manufacturer’s protocol in 6-well or 12-well plates.

Techniques: Hi-C, Control

Journal: Nature communications

Article Title: Stress-induced RNA-chromatin interactions promote endothelial dysfunction.

doi: 10.1038/s41467-020-18957-w

Figure Lengend Snippet: Fig. 4 Inhibition of LINC00607 attenuates SERPINE1 induction and EC dysfunction. a Illustration of LINC00607 genomic locus and gene structure and LNA GapmeRs targeting of LINC00607 RNA. b HUVECs were transfected with scramble (scr) or two LNAs targeting LINC00607. LINC00607 RNA and SERPINE1 mRNA levels were quantified. Data represent mean ± SEM from seven independent experiments. c qPCR of LINC00607 in subcellular fractionations of HUVECs transfected with scr, LNA1, or LNA2. Data represent mean ± SEM from four independent experiments. d RNA-seq was performed with cells transfected as in b in biological replicates. Heatmap is plotted based on z-scaled log-transformed gene expression levels. e, f ECs transfected with scr or LNA1 and then treated by H + T were used in e monocyte adhesion assay and in f SA-β-gal assay. In e, the number of attached monocytes to ECs were quantified. Data represent mean ± SEM from eight independent experiments performed with peripheral blood-derived monocytes from four individual donors. Representative images show the attachment of fluorescently labeled peripheral blood-derived monocytes to ECs for experiments performed with monocytes from three different donors. In f, ECs with positive β-gal staining were quantified. The positively stained cell number in scr control was set to 100 (%). f Data represent mean ± SEM from four independent experiments respectively. Scale bar = 200 μm. * denotes p = 0.0289, 0.0013, 0.001, 0.0001, 0.0025, and 0.024 from left to right (in b) and p = 0.0023, 0.0079, and 0.0249 from left to right (in c) between indicated groups based on ANOVA followed by Bonferroni post hoc test (in b and c); p = 0.0039 (in e) and 0.0014 (in f), as compared to scr group based on two-tailed paired t test (in e and f). Source data are provided as a Source data file.

Article Snippet: Plasmid transfection was performed using the CytofectTM HUVEC Transfection kit (Cell Applications) following the manufacturer’s protocol in 6-well or 12-well plates.

Techniques: Inhibition, Transfection, RNA Sequencing, Transformation Assay, Gene Expression, Cell Adhesion Assay, Derivative Assay, Labeling, Staining, Control, Two Tailed Test