Fig. 2.

PRKAA1/AMPKα1 stimulates the metabolic alteration of ECs in vitro. a Western-blot analysis and quantification data of protein levels of Slc2a1 and Pfkfb3 in MAECs isolated from Prkaa1^f/f and Prkaa1^VEC-KO mice under 25% EGM-2 and VEGF 20 ng/ml 12 h treatment. n = 4. b Western-blot analysis and quantification data of protein levels of SLC2A1 and PFKFB3 in HUVECs transfected with siCtrl and siPRKAA1 under 25% EGM-2 and VEGF 20 ng/ml 12 h treatment. n = 5. c, d Representative images of immunofluorescence staining for cellular Slc2a1 and Pfkfb3 in MAECs isolated from Prkaa1^f/f and Prkaa1^VEC-KO mice under 25% EGM-2 and VEGF 20 ng/ml 12 h treatment. Scale bar: 10 µm; n = 4. e ECAR profile showing glycolytic function and quantification of glycolytic function parameters in MAECs isolated from Prkaa1^f/f and Prkaa1^VEC-KO mice under 25% EGM-2 and VEGF 20 ng/ml 12 h treatment. Vertical lines indicate the time of addition of glucose (10 mM), oligomycin (1 μM), and 2-DG (50 mM). n = 12–16 for each treatment group, replicated four times. f Intracellular lactate levels in MAECs isolated from Prkaa1^f/f and Prkaa1^VEC-KO mice under 25% EGM-2 and VEGF 20 ng/ml 12 h treatment. n = 6. g Representative images and quantification data of the flow cytometry analysis of 2-NBDG (100 µM, 30 min) staining in HUVECs transfected with siCtrl and siPRKAA1 for 48 h. n = 5. h Measurement of intracellular G-6-P, pyruvate and lactate levels in HUVECs transfected with siCtrl and siPRKAA1 for 48 h. n = 5. All data were expressed as mean ± SEM. Statistical significance was determined by unpaired Student’s t-test (for f, g, h) and one-way ANOVA followed by Bonferroni test (for a, b, e). *p < 0.05 was considered significant, **p < 0.01, ***p < 0.001