Fig. 4 | Nature Communications

Fig. 4

From: Endothelial adenosine A2a receptor-mediated glycolysis is essential for pathological retinal angiogenesis

Fig. 4

ADORA2A regulates HRMEC proliferation, sprouting and tube formation. a, b Bromodeoxyuridine (BrdU) staining of HRMECs transfected with siRNAs targeting human ADORA2A (siA2AR) or with a non-targeting negative control (siCtrl) under hypoxia conditions (0.5% O2). n = 6. *P < 0.05. c Cell proliferation measured by WST-1 cell proliferation assay. n = 6. **P < 0.01. d Growth curves of transfected cultures. n = 6. *P < 0.05, ***P < 0.001 vs. siCtrl. eg Ki-67 and BrdU staining of HRMECs. HRMECs were infected with a recombinant adenovirus vector expressing human ADORA2A (Ad-A2AR) or a negative control adenovirus (Ad-Ctrl) in the presence of adenosine. Scale bars: 50 μm. n = 6. ***P < 0.001. h Cell proliferation measured by WST-1 cell proliferation assay. n = 6. ***P < 0.001. i Growth curves of HRMECs over 72 h following infection with Ad-Ctrl or Ad-A2AR in the presence of adenosine. n = 6. ***P < 0.001 vs. Ad-Ctrl. ju HRMECs were transfected with siA2AR or siCtrl, or infected with Ad-Ctrl or Ad-A2AR, and then were cultured in collagen gel to grow into 3D multicellular spheroids, or on a 2D matrix to form a tube network in the presence or absence of VEGF or adenosine. j, m Representative images of spheroidal sprouting after culturing for 24 h in collagen matrix under hypoxia (0.5% O2) or normoxia (21% O2). Scale bars: 100 μm. Morphometric quantification of spheroid sprouting by calculating the number of sprouts per spheroid k, n as well as total sprout length l, o. n = 10 per group. n is number of spheroids quantified. **P < 0.01; ***P < 0.001. Representative fluorescence photographs of angiogenic tube formation p, s. Scale bars: 200 μm. Cumulative tube length quantified using the Image J software q, t, and branch points calculated from five experiments in each case r, u. *P < 0.05; **P < 0.01; ***P < 0.001. Data are represented as means ± s.e.m. Statistical significance was determined by unpaired Student’s t-test (for b, c, f, g, h, k, l, n, o, q, r, t, u) and two-way ANOVA followed by Bonferroni test (for d, i)

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