Journal: Nature Cardiovascular Research

Article Title: Single-cell RNA sequencing reveals sex differences in the subcellular composition and associated gene-regulatory network activity of human carotid plaques

doi: 10.1038/s44161-025-00628-y

Figure Lengend Snippet: The top key drivers of GRN195, PLVAP and FAM110D , were overexpressed in HAECs followed by RNA-seq. a , PLVAP and FAM110D are highlighted in the color-coded GRN195 network according to the cell type specificity. b , Average pair-wise correlation of GRN195 coding genes across different conditions, comparing Mock, oePLVAP and oeFAM110D groups with four replicates each. Mock, cells with lenti virus without construct, i.e., acts as control; oePLVAP, primary cells transduced with lentiviral vectors to overexpress PLVAP gene; oeFAM110D, primary cells transduced with lentiviral vectors to over express FAM110D gene; cor.cor, concordance of correlation structure. In the box plot, the center line denotes the median value (50th percentile), and the box contains the 25th to 75th percentiles of the dataset. The statistical test was conducted using t -test (two-sided). No adjustment was made and there was no multiple comparison. c , GSEA of RNA-seq data of HAECs overexpressing PLVAP and FAM110D . The NES indicate the direction and strength of gene set enrichment. The statistical test used was GSEA, which uses a one-sided test to assess whether a predefined gene set is significantly enriched at either the top or bottom of the ranked gene list. P values were calculated using permutation testing, and multiple comparisons were adjusted using the FDR correction. d , Relative proliferation in the PLVAP group compared with the control (CTL) group shows a significant increase, while the FAM110D group shows a significant decrease relative to CTL. Statistical analysis was conducted using repeated-measures ANOVA (two sided) Dunnett’s multiple comparison test, P < 0.0001. Data represent the mean ± s.d. ***, P < 0.0001. e , Representative images of cell painting showing nuclear (blue), mitochondria (red) and nucleic acid (yellow) stains at day 6 in LV-overexpressing HAEC cells. Scale bar = 50 µm. A total of 4 wells per group including 10 regions per well were imaged ( n = 40 images per group) with two biological replicates. f , Heatmap illustrating the effect of LV- FAM110 and LV- PLVAP overexpression on cell phenotypic features in HAECs at day 6 detected by cell painting. A total of 4 wells per group including 10 regions per well were imaged ( n = 40 images per group) with Opera Phenix Plus High Content Screening System, and the cell morphological signature was analyzed by Harmony analysis software. Two independent experiments were performed. Heatmap values show the fold changes over the LV-EGFP control vector. mito, mitochondria.

Article Snippet: After washing three times with DPBS, the plates were imaged by using Opera Phenix Plus High Content Screening System (Revvity) with the ×40 water immersion objective and excitation and emission wavelengths as previously recommended .

Techniques: RNA Sequencing, Virus, Construct, Control, Transduction, Comparison, Over Expression, High Content Screening, Software, Plasmid Preparation