Fig. 2
ECFC responses to 1% O2. ECFCs were grown in 12% FCS-supplemented EBM-2 medium and maintained in either 21 or 1% O2 for the indicated times. a Protein lysates isolated from time-matched ECFCs grown under normal conditions (21% O2) or hypoxic conditions (1% O2) were probed with HIF-1α (top panel) or HIF-2α, with β-actin included as a loading control (bottom panel). b. ECFCs cultured at 21 or 1% for 5 h were permeabilised and incubated with antibodies reactive to HIF-α (red) and DAPI to stain the nuclei (blue). Scale bar: 50 μM. c Real-time PCR was carried out on cDNA generated from ECFCs incubated in 1% O2 for the indicated times. Levels of HIF-1 responsive genes ANGPTL4 and SLC2A1 were measured, and normalised to the mean of two housekeeping genes (18S and β-actin). The ∆∆Ct subtractive method was used to compare between treatments, with 21% O2 as the calibrator. Fold-changes compared with 21% O2 were calculated. Data plotted as mean fold-change ± SD. *p < 0.05 (n = 5). HIF-1α hypoxia inducible factor 1 alpha (Colour figure online)