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.