Fig 1.
Increasing doses of Allopurinol reduce HIF-1α levels in normoxic HFFs and HUVEC cells.
A. Characterisation of HFF and HUVEC response to hypoxia. Cells were exposed to hypoxia (1% oxygen) for the indicated periods of time. At the end of incubation, protein levels were determined in whole cell extracts by immunoblot analysis using the depicted antibodies. B. Cells were treated with Allopurinol at 10, 100 and 1000 μg/ml for 17 hours. Then the cells were lysed for assessment of the indicated protein levels. Cells were treated with Etoposide (Etop) for 24 hours under normoxia. H-cells exposed to 1% O2 for 16 hours. HIF-1α levels were quantified using ImageJ software and graph depicts mean and standard deviation of a minimum of three independent experiments. Anova t-test was performed and p values calculated as follows: *p<0.05; **p<0.01; ***p<0.001.
Fig 2.
Increasing doses of Allopurinol reduce HIF-1α levels in hypoxic HFFs, without changing HIF mRNA levels but have reduced effect in HUVEC cells.
A. Cells were pre-treated with Allopurinol at 10, 100 and 1000 μg/ml for 5, 30 and 60 minutes then were incubated in 1% oxygen for 16 hours. The cells were lysed for assessment of the indicated proteins. HIF-1α levels were quantified using ImageJ software and graph depicts mean and standard deviation of a minimum of three independent experiments. Anova t-test was performed and p values calculated as follows: *p<0.05; **p<0.01; ***p<0.001. B. HIF-1α and HIF-2α mRNA levels were analysed by quantitative PCR in HFF and HUVEC cells. Graph depicts the mean and standard deviation of a minimum of three independent experiments performed in duplicate. Anova t-test was performed and p values calculated as follows: *p<0.05; **p<0.01; ***p<0.001.
Fig 3.
Allopurinol reduces HIF-1α levels independent of PHD function.
A. Cells were pre-treated with Allopurinol at 10 and 1000 μg/ml for 60 minutes and then were incubated in 1% oxygen or treated with 200 μM DFX for 16 hours. Whole cell lysates were analysed by immunoblot using the indicated antibodies. B. Cells were pre-treated with Allopurinol at 1000 μg/ml for 60 minutes and then were incubated with 20μM MG132 for 3 hours. Whole cell lysates were analysed by immunoblot using the indicated antibodies. HIF-1α levels were quantified using ImageJ software and graph depicts mean and standard deviation of a minimum of three independent experiments. Anova t-test was performed and p values calculated as follows: *p<0.05; **p<0.01; ***p<0.001.
Fig 4.
Allopurinol alters angiogenic traits of HUVEC in an in vitro endothelial tube model.
Cells were treated with Allopurinol at 0, 10, 100, 1000 μg/ml, and analysed at 24 hours. A, control group without Allopurinol. B, 10 μg/ml Allopurinol. C, 100 μg/ml Allopurinol. D, 1000 μg/ml Allopurinol. Graph depicts mean and standard deviation of tube length measures in a minimum of three independent experiments performed in triplicate. Anova t-test was performed and p values calculated as follows: *p<0.05; **p<0.01; ***p<0.001.