Figure 3

Effects of the inhibition of glycolysis or mitochondrial respiration on survival of endothelial tip cells and non-tip cells and on proliferation of ECs in HUVEC cultures. (a) Apoptotic cell death of tip cells and non-tip cells after flow cytometric analysis of HUVECs stained with both PE-conjugated anti-CD34 antibody and FITC-conjugated annexin V antibody at 72 h after siRNA transfections against PDHA1, PFKFB3, or LDHA, respectively. Four quadrants (Q) representing: viable tip cells (Q1), apoptotic tip cells (Q2), apoptotic non-tip cells (Q3), and viable non-tip cells (Q4). (b) Inhibition of PDHA1 expression increased the percentage of apoptotic tip cells, whereas no effect was found in non-tip cells. Inhibition of PFKFB3 or LDHA expression, respectively, did not alter percentages of apoptotic tip cells and non-tip cells. (c) Cell proliferation assay of HUVECs expressed as percentages of non-proliferating cells in the G0/G1 phase, and proliferating cells in early S phase, late S phase and G2M phase after flow cytometric analysis of fluorescence of incorporated EdU at 72 h after siRNA transfections against PDHA1, PFKFB3, or LDHA, respectively. (d) Inhibition of the expression of PDHA1, PFKFB3, or LDHA lowered percentages of proliferating HUVECs in the late S phase and G2M phase. Results are shown as means ± SEM of experiments with HUVECs of at least 3 donors. *P < 0.05, **P < 0.01, and ***P < 0.001 as compared to siNT (Unpaired Student’s t-test).