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. Author manuscript; available in PMC: 2012 Aug 16.

Published in final edited form as: Exp Cell Res. 2010 Mar 19;316(10):1706–1715. doi: 10.1016/j.yexcr.2010.03.005

A. To test the ability of HIF-1α to mediate downstream gene transcription under the influence of 12-LOX, nL-8 and neo cells were transiently co-transfected with the 5xHRE and the lacZ expression vectors (see methods). 48 h post transfection, cells were incubated under hypoxic conditions for 6 h. Results are luciferase activity normalized to β-gal activity represented as mean from n=3 ± SD; * represents p-value assessed from student s t-test. B. nL-8 and neo cells were transiently transfected with either an siRNA construct targeted against HIF-1α or a scrambled construct. 48 h post transfection, the cells were incubated under hypoxic conditions for 6 h. Protein samples harvested from the incubated cells were tested for the levels of HIF-1α by immunoblotting. Shown here is a representative result from three independent experiments. C. Conditioned media from the HIF-1α siRNA and the control siRNA transfected cells were assayed for the levels of secreted VEGF using a VEGF ELISA kit according to the manufacturer s instructions. Values were normalized to the total number of cells. Shown here is a representative result from three independent experiments. D. nL-8 and neo cells were co-transfected with either a full length VEGF promoter vector (p1176) or a VEGF promoter vector with the HRE region deleted (p888) and a lacZ vector. 48 h post transfection, cells were washed and incubated under hypoxic conditions for 6 h, lysates were tested for luciferase and β-gal activities. Shown here are the normalized luciferase values from three independent experiments performed under essentially identical conditions. Results are expressed as mean of n=3 ± SD; * represents p value assessed using student s t-test.

A. To test the ability of HIF-1α to mediate downstream gene transcription under the influence of 12-LOX, nL-8 and neo cells were transiently co-transfected with the 5xHRE and the lacZ expression vectors (see methods). 48 h post transfection, cells were incubated under hypoxic conditions for 6 h. Results are luciferase activity normalized to β-gal activity represented as mean from n=3 ± SD; * represents p-value assessed from student s t-test. B. nL-8 and neo cells were transiently transfected with either an siRNA construct targeted against HIF-1α or a scrambled construct. 48 h post transfection, the cells were incubated under hypoxic conditions for 6 h. Protein samples harvested from the incubated cells were tested for the levels of HIF-1α by immunoblotting. Shown here is a representative result from three independent experiments. C. Conditioned media from the HIF-1α siRNA and the control siRNA transfected cells were assayed for the levels of secreted VEGF using a VEGF ELISA kit according to the manufacturer s instructions. Values were normalized to the total number of cells. Shown here is a representative result from three independent experiments. D. nL-8 and neo cells were co-transfected with either a full length VEGF promoter vector (p1176) or a VEGF promoter vector with the HRE region deleted (p888) and a lacZ vector. 48 h post transfection, cells were washed and incubated under hypoxic conditions for 6 h, lysates were tested for luciferase and β-gal activities. Shown here are the normalized luciferase values from three independent experiments performed under essentially identical conditions. Results are expressed as mean of n=3 ± SD; * represents p value assessed using student s t-test.