Fig. 8 | Nature Communications

Fig. 8

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

Fig. 8

ADORA2A activation mediates increase in glycolysis via a HIF-1α-dependent pathway. ad Western blot analysis of HIF-1α mRNA and protein expression in HRMECs transfected with siA2AR or siCtrl under hypoxia (0.5% O2) or normoxia (21% O2) a, b, or infected with Ad-Ctrl or Ad-A2AR under normoxia c, d. n = 4. ***P < 0.001. e, f HIF-1α immunofluorescent staining of OIR retinas from Adora2a WT and Adora2a VEC-KO mice at postnatal day (P)15. Representative green (HIF-1α), red (CD31), blue (nuclei, DAPI), and merged images captured with confocal fluorescent microscopy. GCL ganglion cell layer, INL inner nuclear layer, ONL outer nuclear layer. Scale bar, 50 μm. The fluorescence intensity of HIF-1α staining was calculated by Image J software then normalized to that of WT control (Adora2a WT). n = 6 mice for each group. **P < 0.01. g Real-Time PCR analysis of the mRNA levels of glycolytic genes in HRMECs transfected with siHIF-1α or siCtrl under normoxia (21% O2) or hypoxia (0.5% O2). n = 3. *P < 0.05; **P < 0.01; ***P < 0.001 vs. siCtrl normoxia group; & P < 0.05; && P < 0.01 vs. siCtrl hypoxia group. h Real-Time PCR analysis of the mRNA levels of glycolytic genes in HRMECs. Cells were transfected with HIF-1α siRNA or siCtrl for 24 h under normoxia, and further infected with Ad-A2AR or Ad-Ctrl for an additional 24 h, followed by adenosine treatment for another 12 h. n = 4. *P < 0.05; **P < 0.01; ***P < 0.001 vs. Ad-A2AR + Ado + siCtrl group. i Quantification of glycolytic function in HRMECs transfected with siCtrl or siHIF-1α in the presence of CoCl2 (200 μM). n = 8 per group. *P < 0.05; ***P < 0.001. j Quantification of glycolytic function in HRMECs infected with Ad-Ctrl or Ad-A2AR, with or without adenosine treatment, in the presence or absence of the HIF-1α inhibitor CAY10585. n = 7, 7, 6, respectively. *P < 0.05; ***P < 0.001. Data are represented as means ± s.e.m. Statistical significance was determined by unpaired Student’s t-test (for d, f, g, h) and one-way ANOVA followed by Bonferroni test (for b, i, j)

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