Fig 1: CD97 promotes membrane ruffling and lamellipodia formation in endothelial cells. (A) Validation of the CD97 knockout status of endothelial cells generated by clustered regularly interspaced short palindromic repeats/Cas9 using western blotting. (B) Alterations in the distribution of F-actin in endothelial cells, CD97-Cas9 endothelial cells or CD97-lentivirus endothelial cells. Stress fibers or lamellipodia are indicated by white arrows. Scale bar, 3 µm. (C) Protein levels of RHO, RAC and CDC42 in endothelial cells, CD97-Cas9 endothelial cells or CD97-lentivirus endothelial cells. (D) Analysis of protein expression levels of CD97 in endothelial cells, CD97-lentivirus endothelial cells or CD97-lentivirus endothelial cells transfected with siRNA to knockdown the expression of CDC42. CD97, cluster of differentiation 97; Cas9, clustered regularly interspaced short palindromic repeats-associated protein9; Rho, Ras homolog; Rac, Ras-related C3 botulinum toxin substrate; Cdc42, cell division cycle 42; Arp2, actin-related protein 2; NC, negative control; si, small interfering RNA; leti, lentivirus.
Fig 2: CD97 alters the GBM immune microenvironment. Compared to CD97lo GBMs, CD97hi GBM tumors exhibited significantly greater proportion of (A) nonpolarized M0 macrophages, (B) regulatory T cells, and (C) resting NK cells. In contrast, compared to CD97hi GBM tumors, CD97lo GBM tumors had higher proportions of (D) monocytes. (E) THP-1 monocytes co-cultures with wild type U251 GBM cells had significantly higher macrophage differentiation compared to those co-cultured with U251 CD97-knockdown cells (P < 0.001). *P < 0.05; **P < 0.01; ***P < 0.001.
Fig 3: Single-cell analysis of CD97-expressing glioblastoma cells. Glioblastoma (GBM) cells from 14 IDH-wild type cases were subjected to single-cell analysis. (A) Shown is a t-SNE plot classifying cells from these 14 cases into 19 clusters. (B) Neoplastic versus non-neoplastic cells were identified and highlighted in pink via copy number variation (CNV) analysis, with stromal cells shown in cyan. (C) Neoplastic cells (n = 2530) were clustered into 19 distinct cell populations based off differential gene expression. (D) CD97+ and CD97− cells are highlighted among neoplastic cells from these 19 clusters from 14 GBM patient samples. (E) Percentage of CD97+ cells among the 19 clusters derived from 2,530 tumor cells.
Fig 4: Overexpression of CD97 reverses the dysregulation of high glucose-induced endothelial cell migration. (A) Confirmation of the overexpression of CD97 in endothelial cells transfected with a lentivirus using western blot analysis. (B) Representative images from wound healing assays prior to (0 h) and following (24 h) high glucose (33 mM) or basal glucose (5.5 mM; control) treatment of normal and CD97-lentivirus endothelial cells. Scale bar, 70 µm. CD97, cluster of differentiation 97; HG, high glucose; leti, lentivirus; HG, high glucose.
Fig 5: High glucose concentrations inhibit CD97 transcription via the upregulation of STAT1. (A) Schematic representation of the promoter regions (upper), which were sub-cloned into the pGL3-basic luciferase reporter. Activation of the promoter-luciferase reporters in response to high glucose concentrations in endothelial cells is shown below. (B) Top five most robust binding transcription factors in the CD97 promoter region, determined using TF filter plate assays. (C) High glucose (33 mM) stimulation promoted STAT1 nuclear translocation; 5.5 mM glucose stimulation was used as a control. (D) Analysis of the mRNA transcript expression of CD97 in endothelial cells transfected with siRNA targeting STAT1 prior to high glucose (33 mM) treatment (5.5 mM glucose served as a control). (E) Schematic illustration of potential binding sites in the CD97 promoter for STAT1. High glucose (33 mM) treatment increased STAT1 binding to its binding site in the CD97 promoter (5.5 mM glucose served as a control). *P<0.05. CD97, cluster of differentiation 97; ADGRE5, adhesion G protein-coupled receptor 5; STAT1, signal transducer and activator of transcription 1; NF1, neurofibromatosis type 1; SP1, specificity protein 1; PAX5, paired box 5; GATA, GATA binding protein; HG, high glucose; si, small interfering RNA.
Supplier Page from Abcam for Anti-CD97 antibody [EPR4427]