Fig. 4

β-catenin increases the expression of KDR by upregulating NANOG under AGEs treatment. A, B 293T cells were transfected with KDR promoter luciferase construct followed by AGEs stimulation in the presence or absence of 20 μM ICG-001 for 24 h (A) or β-catenin siRNA (B). The Dual-Luciferase Reporter Assay was used to detect promoter activity, and luminescence was normalized to Renilla. n = 5. C 293T cells were co-transfected with KDR promoter luciferase construct and β-catenin-overexpression plasmid. After 24 h. The Dual-Luciferase Reporter Assay was performed to detect promoter activity, and luminescence was normalized to Renilla. n = 5. D Schematic diagram of the predicted binding region of β-catenin to the KDR promoter DNA sequence (upper panel) and the PCR amplification product location of 8 primers for the predicted binding element (lower panel). IBS software was used as a drawing tool [32]. E The binding of β‐catenin to the KDR promoter in response to AGEs treatment was determined in HUVECs by ChIP assay and quantified by PCR. Experiments were repeated three times with similar results and one representative result is shown. F Venn diagram showing overlapping genes between transcriptional factors of KDR and Wnt/β-catenin target genes. G-I HUVECs were treated with AGEs (100 μg/ml) for different times and the mRNA levels of GATA2/SP1/NANOG were detected by qPCR. n = 5 to 7. J, K HUVECs were pretreated with ICG-001 (20 μM) (J) or β-catenin siRNA (K) followed by AGEs (100 μg/ml) stimulation and NONOG mRNA levels were detected by qPCR. n = 5. L siRNA targeting NANOG was transfected into HUVECs followed by AGEs (100 μg/ml) treatment and KDR mRNA levels were detected by qPCR, respectively. n = 4. M 293T cells were co-transfected with KDR promoter luciferase construct, β-catenin-overexpression plasmid and NONOG siRNA. After 48 h, the Dual-Luciferase Reporter Assay was conducted to detect KDR promoter activity, and luminescence was normalized to Renilla. n = 6. Data are shown as Mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001