Figure 3.

Notch is a metabolic brake for G₁ cell cycling in ECs and overexpression of PFKFB3 overcomes the Notch-mediated metabolic brake. For all panels, G₀-synchronized ECs (obtained by contact inhibition) were replated and analyzed at the indicated time points. (A) Time course FACS analysis using Hoechst33342 and PyroninY in control (gray) and Dll4-treated ECs (red). Around 18 hours after reseeding, control cells entered S phase. By contrast, fewer Dll4-treated cells entered S phase (average of >10 experiments, each with triplicate measurements; data are mean ± SEM). ** P < 0.01, *** P < 0.001 versus control at the corresponding time point; ### P < 0.001 vs. corresponding baseline (14 hours). Statistics: mixed model statistics considering experiment as co-variant. (B) Time course analysis of glycolytic flux. Around 16 hours after reseeding and beyond, glycolytic flux was increased in control cells but only minimally in Dll4-treated cells (average of 5 experiments, each with triplicate measurements; data are mean ± SEM). * P < 0.05, ** P < 0.01, *** P < 0.001 versus control at the corresponding time point; ### P < 0.001 vs. corresponding baseline (14 hrs); Statistics: mixed model statistics considering experiment as co-variant. (C) Kinetic comparison of metabolic and cell cycle changes after replating of the G₀-synchronized cells. Plotting the control data shown in panel A and B together illustrates that the metabolic change (full line) shows an increase preceding (16 hrs, arrow) that of the cell cycle change (dashed line) (average of 5 experiments, each with triplicate measurements; data are mean ± SEM). ### P < 0.001 versus corresponding baseline (14 hrs); Statistics: mixed model statistics considering experiment as co-variant. (D) Representative immunoblot of PFKFB3 for control and PFKFB3^OE cells. β-actin is used as loading control. (E) Glycolytic flux, showing enhanced glycolysis upon PFKFB3 overexpression (PFKFB3^OE) in control conditions (without Dll4). Dll4 lowers glycolysis, but PFKFB3^OE enhances glycolysis again in Dll4-activated cells. (F) [³H]-thymidine incorporation in DNA, showing that PFKFB3^OE increases EC proliferation. Dll4 reduces EC proliferation, but PFKFB3^OE increases again the proliferation of Dll4-activated ECs. Data in E and F are mean ± SEM of n = 4 independent experiments, each performed in triplicate. *P < 0.05, **P < 0.01, ***P < 0.001 by mixed model statistics considering experiment as co-variant.