Figure 2. HIF-1α drives expression of
chordin-like 1 in retinal pericytes exposed to hypoxia. A:
western blot analysis of nuclear extracts generated from human retinal
pericytes exposed to increasing periods of hypoxia (0, 6, 24, and 48 h)
for HIF-1α shows upregulation of the protein by 6 h. B:
Transfection of human retinal pericytes maintained in normoxia with
expression vectors for HIF-1 α, C is control/empty vector, WT is wild
type vector, WT-HIF-1 α, DM is double mutant vector, DM-HIF-1 α, using
the transfection reagent Fugene6, induces expression of many of the
genes upregulated in response to hypoxia, as measured by RT–PCR. 18S
PCR is shown as a loading control and western blot analysis confirmed
expression from each of the HIF-1α expressing plasmids. C:
Induction of CHL-1 mRNA in response to HIF-1α overexpression
was quantitated by real time PCR. CHL-1 levels were normalized
to 18S rRNA, using a pre-developed assay reagent. Data are expressed as
mean relative quantity of mRNA, to control, for three independent
experiments ±standard error of measurement. D: HeLa cells were
transfected, using the transfection reagent Fugene6, with the CHL-1
promoter or a luciferase reporter construct containing four HIF-1α
responsive elements (HRE), alone (control), cotransfected with the
HIF-1α expression vector DM-HIF-1α, or alone and subsequent exposure to
hypoxia for 24 h (1% O2). Cotransfection with DM-HIF-1α as
well as exposure to hypoxia induced activation of the CHL-1 promoter
and the HRE construct. Data are expressed as mean±SEM values. The
asterisk indicates a significance at p<0.05 and the double asterisk
indicates a significance at p<0.001.