Additional file 1 of Down-regulation of WWP2 aggravates Type 2 diabetes mellitus-induced vascular endothelial injury through modulating ubiquitination and degradation of DDX3X
Creators: name: You, Shilong
name: Xu, Jiaqi
name: Yin, Zeyu
name: Wu, Boquan
name: Wang, Pengbo
name: Hao, Mingjun
name: Cheng, Cheng
name: Liu, Mengke
name: Zhao, Yuanhui
name: Jia, Pengyu
name: Jiang, Hongkun
name: Li, Da
name: Cao, Liu
name: Zhang, Xingang
name: Zhang, Ying
name: Sun, Yingxian
name: Zhang, Naijin
Publisher: name: figshare
Year: 2023
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Molecular Biology
subject: Physiology
subject: FOS: Biological sciences
subject: Chemical Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Virology

description: Additional file 1: Fig. S1 Single-cell analysis of endothelial cells from healthy and T2DM donors. A, B UMAP plot showing 6,917 mesenteric arterial cells isolated from 2 healthy control (N) and 1 T2DM (T) male donors. Number of cells sequenced are: N1_1: 1069, N1_2: 729, N2_1: 389, N2_2: 382, N2_3: 406, N2_4: 306, T2_1: 1814, T2_2: 1822. C UMAP plot showing clusters identified by graph-based semi-unsupervised clustering. D Dot plot of top five traditional markers used for cell identity. E, F Feature plots showing gene expression of Cdh5 (E) and Pecam1 (F), as representative genes of classical markers for endothelial cells. Ctrl, control; T2DM, Type 2 diabetes mellitus; UMAP, Uniform Manifold Approximation and Projection. Fig. S2 WWP2-mediated Ubiquitin (Ub)-dependent degradation of DDX3X is not a lysosome pathway. A Effects of lysosome inhibitor chloroquine (CQ) on WWP2-mediated K63-linked polyubiquitination of DDX3X. Human umbilical vein endothelial cells (HUVECs) were transfected with expression plasmids encoding HA-WWP2 and Ub K63, and then treated with the indicated concentrations of CQ for 24 h or DMSO as control. B, C Representative Western blotting analysis (B) and quantitative analysis (C) of HA-WWP2 and DDX3X protein levels. HUVECs were transfected with expression plasmids encoding HA-WWP2, and then treated with the indicated concentrations of CQ for 24 h or DMSO as control. Values are shown as mean ± SD (***P &lt; 0.001, one-way ANOVA with Dunnett’s multiple comparison post-hoc test). Fig. S3 Endothelial-specific Wwp2 knockout in mice leads to up-regulation of endothelial DDX3X expression. A, B, C, D Representative immunofluorescent staining of aortic cross sections for the indicated mice (A, C) and quantitative analysis of relative fluorescence intensity of DDX3X in CD31 positive cells (B, D). (n = 6 mice per group). Red, DDX3X; Green, CD31 (a marker of ECs); blue, DAPI. scale bar 50 µm. (***P &lt; 0.001, two-tailed unpaired Student t-tests). Cdh5 Cre-, (Cdh5 Cre-; Wwp2fl/fl mice); Cdh5 Cre+, (Cdh5 Cre+; Wwp2fl/fl mice); DAPI, 4′,6-diamidino-2-phenylindole; ECs, Endothelial cells; T2DM, Type 2 diabetes mellitus. Table S1. The clinical information of donors in the GSE156341 dataset. BMI, Body Mass Index; HC, healthy control; T2DM, Type 2 diabetes mellitus. Table S2. Cell types_top50_markers. Cell types were identified according to the expression of top 50 known markers. Table S3. Expression changes of genes in KEGG apoptosis pathway. NS, not significant.