Fig. 7 | Nature Communications

Fig. 7

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

Fig. 7

ADORA2A regulates HRMEC tip cell formation. a Real-Time PCR analysis of the mRNA levels of the sprouting-governing genes in HRMECs transfected with siA2AR or siCtrl, in the presence or absence of DAPT (n = 3; *P < 0.05; **P < 0.01; ***P < 0.001 vs. siCtrl; & P < 0.05, && P < 0.01 vs. siCtrl + DAPT). b Real-Time PCR analysis of the mRNA levels of the sprouting-governing genes in 2-DG-treated HRMECs in the presence or absence of DAPT (n = 4; *P < 0.05; **P < 0.01; ***P < 0.001 vs. vehicle control; & P < 0.05, && P < 0.01 vs. DAPT). c, d Morphometric quantification of spheroid sprouting from cells transfected with siCtrl or siA2AR in the presence or absence of DAPT. n = 10 per group. n is number of spheroids quantified. *P < 0.05; **P < 0.01; ***P < 0.001. e Representative fluorescence photographs of EC spheroids containing a 1:1 mixture of siCtrlGFP and siCrlRED ECs, or a 1:1 mixture of siCtrlGFP ECs and ECs with siA2ARRED in the presence of DAPT. Scale bars, 100 μm. f Quantification of the fraction of tip cells with the indicated genotypes shown in e (n = 10; ***P < 0.001 vs. siCtrlGFP). g Representative fluorescence photographs of mosaic EC spheroids containing a 1:1 mixture of siCtrlGFP ECs and siA2ARRED ECs under hypoxia conditions. Cells were stained with DRAQ5 (blue) to mark EC nuclei. Tip cells are indicated by “^“; Stalk cells are indicated by “*“. The 2nd and 4th rows are magnification of the boxed regions in the 1st and 3rd rows, respectively. Scale bar: 50 μm (1st and 3rd rows) and 20 μm (2nd and 4th rows). h Quantification of the fraction of tip cells with the indicated genotypes shown in g (n = 10 per group). ***P < 0.001 vs. siCtrlGFP. Data are represented as means ± s.e.m. Statistical significance was determined by unpaired Student’s t-test (for a, b, f, h) and one-way ANOVA followed by Bonferroni test (for c, d)