Figure 6

Figure 6

Inhibition of autophagy activates p53 and induces apoptosis in endothelial cells under hypoxia and nutrient deprivation. A and B: Immunofluorescent staining of HUVECs cultured under different conditions: control, hypoxia (1% O₂), starvation (glucose and serum starvation in MEM), and combined hypoxia and starvation (HS). LC3B (red) marks autophagic activity, whereas DAPI (blue) stains the nuclei. Quantification of LC3B puncta-positive cells showed a significant increase in autophagy under combined hypoxia and nutrient deprivation compared to that in the control (****P < 0.0001). Scale bar = 50 μm. C: Volcano plot displaying DEGs in HUVECs cultured under hypoxia and nutrient deprivation versus controls. Upregulated genes are highlighted in red, downregulated genes in blue, and stable genes in grey. MAP1LC3B2 expression was significantly upregulated, indicating elevated autophagic activity. D: GO analysis showing the enriched pathways in HUVECs cultured under hypoxia and nutrient deprivation. E: Volcano plot comparing gene expression in HUVECs treated with hypoxia and nutrient deprivation combined with 4 μmol/L ULK-101 (autophagy inhibitor) versus hypoxia and nutrient deprivation alone. Apoptosis-related genes, including FADD, BAX, CASP9, and FAS, were significantly upregulated, while TP53 expression was restored by ULK-101 treatment. F: GO analysis of HUVECs treated with ULK-101 under hypoxia and nutrient deprivation conditions, showing significant enrichment of apoptosis-related pathways, angiogenesis, and positive regulation of immune responses. G and H: Flow cytometry analysis of apoptosis in HUVECs under control, hypoxia and nutrient deprivation (HS), 4 μmol/L ULK-101, and combined ULK-101 and HS treatments. Apoptosis was measured using Annexin V/PI staining. Quantification (H) showed a significant increase in apoptosis in HUVECs treated with ULK-101 under hypoxic and nutrient-deprived conditions (****P < 0.0001; **P < 0.01). I and J: Immunofluorescence staining of HUVECs treated with control, hypoxia and nutrient deprivation (HS), 4 μmol/L ULK-101, and combined ULK-101 and HS treatments, with and without the p53 inhibitor pifithrin-α (PFTα) HBr (10 μmol/L). P53 (red) and cleaved caspase-3 (green) indicate apoptosis, with DAPI staining of the nuclei (blue). Quantification revealed a significant increase in nuclear p53 translocation after ULK-101 treatment (J). Scale bar = 50 μm. K: Western blot analysis of p53 and BAX protein expression in HUVECs under control, hypoxia and nutrient deprivation (HS), 4 μmol/L ULK-101, and combined treatment with 10 μmol/L PFTα. The 4 μmol/L ULK-101 treatment significantly increases p53 and BAX expression under hypoxia and nutrient deprivation.