Journal: Stem Cell Research & Therapy

Article Title: Restoration of NOX4 signalling reverses endothelial colony-forming cell angiogenic dysfunction associated with experimental and clinical diabetes

doi: 10.1186/s13287-025-04393-4

Figure Lengend Snippet: Clinical diabetes induces dysregulation of CB-ECFC angiogenic pathways linked with downstream NOX4-dependent pro-angiogenic signalling. CB-ECFCs were isolated from healthy and gestational diabetic donors and subjected to RNA sequencing prior to ( A ) IPA network generation, node colour represents predicted gene activation (orange) and inhibition (blue). B , C Proteome Profiler™ analysis of pooled lysates (triplicates from 5 clones) from diabetic CB-ECFCs subjected to electroporation for introduction of either empty vector (EV; pcDNA4/TO/myc-His A) or NOX4 overexpression (OE; pcDNA4/TO/NOX4-myc-His A) construct for detection of protein phosphorylation. Differential expression between groups presented as a heat map indicating largely decreased (red) phosphorylation (OE versus EV). D – G Protein expression of endoglin, E2F1 and SERPINE1 by Western blotting with normalisation to ACTB as loading control; n = 8/9, combined data from 3 different clones. Representative cropped blots shown from one healthy and one diabetic clone. Full-length blots/gels are presented in Supplementary Figure. Data are mean ± SEM; ** P < 0.01, *** P < 0.001, paired Student’s t -test

Article Snippet: Membranes were then incubated overnight at 4 °C with primary antibodies against ACTB (#3700, Cell Signaling Technology, 1:5000), NOX2 (ab80508, Abcam, 1:2000), NOX4 (ab133303, Abcam, 1:2000), VEGF Receptor 2 (55B11, Cell Signaling Technology, 1:1000), Phospho-VEGF Receptor 2 (Try 1175; D5B11, Cell Signaling Technology, 1:1000), eNOS (610297, BD Transduction Labs, 1:500), Phospho-eNOS (S1177, 9571S Cell Signaling Technology, 1:1000), SERPINE1 (66261-1-Ig, Proteintech, 1:10000), Endoglin (#14606, Cell Signaling Technology, 1:1000) and E2F1 (66515-1-Ig, Proteintech, 1:3000).

Techniques: Isolation, RNA Sequencing, Activation Assay, Inhibition, Clone Assay, Electroporation, Plasmid Preparation, Over Expression, Construct, Phospho-proteomics, Quantitative Proteomics, Expressing, Western Blot, Control

Journal: Stem Cell Research & Therapy

Article Title: Restoration of NOX4 signalling reverses endothelial colony-forming cell angiogenic dysfunction associated with experimental and clinical diabetes

doi: 10.1186/s13287-025-04393-4

Figure Lengend Snippet: Restoration of NOX4 expression in diabetic CB-ECFCs rescues angiogenic capacity by specifically inducing pro-angiogenic signalling. Summary schematic indicating that restoration of NOX4 levels in diabetic CB-ECFCs leads to upregulated E2F1 signalling in parallel with fully restored angiogenic function. E2F1 is a transcription factor which positively regulates key genes associated with efficient progression through the cell cycle, DNA replication and subsequent proliferation, which is critical to support an efficient and potent pro-angiogenic response. NOX4 induction in diabetic CB-ECFCs also led to reduced phosphorylation of anti-proliferative P53 (at S46), increased expression of SERPINE1 and endoglin, which are implicated in pro-angiogenic signalling

Article Snippet: Membranes were then incubated overnight at 4 °C with primary antibodies against ACTB (#3700, Cell Signaling Technology, 1:5000), NOX2 (ab80508, Abcam, 1:2000), NOX4 (ab133303, Abcam, 1:2000), VEGF Receptor 2 (55B11, Cell Signaling Technology, 1:1000), Phospho-VEGF Receptor 2 (Try 1175; D5B11, Cell Signaling Technology, 1:1000), eNOS (610297, BD Transduction Labs, 1:500), Phospho-eNOS (S1177, 9571S Cell Signaling Technology, 1:1000), SERPINE1 (66261-1-Ig, Proteintech, 1:10000), Endoglin (#14606, Cell Signaling Technology, 1:1000) and E2F1 (66515-1-Ig, Proteintech, 1:3000).

Techniques: Expressing, Phospho-proteomics