The regulation of Hypoxia-Inducible Factor-1 (HIF-1alpha) expression by Protein Disulfide Isomerase (PDI)
Fig 5
Effects of PHD on HIF-1alpha expression.
(A) Hep3B cells overexpressing PDI were cultured under normoxic conditions, and total RNA was isolated from cells in three different plates. HIF-1alpha mRNA levels were measured by RT-PCR. The HIF-1alpha mRNA levels of mock cells were set to 1.0. (B and C) Hep3B cells overexpressing PDI were cultured for 6 h in the presence of 100 μM dipyridyl (B) or for 16 h in the presence of 0.5 mM DMOG (C) under normoxic conditions, and immunoblotting was performed. The HIF-1alpha protein levels of mock cells in the presence of dipyridyl (B) or DMOG (C) were set to 1.0. (D and E) PDI/pcDNA 3.1 (+) and si-PHD2 (D) or sh-Ref-1 (E) were transfected into Hep3B cells, cells were cultured for 6 h under hypoxic conditions (E). Proteins (3 or 15 μg) were separated by SDS-PAGE, and immunoblotting was performed with the anti-PHD2 or -Ref-1 antibody (1:1000 dilution). The HIF-1alpha protein levels of si-ctrl or sh-ctrl cells under hypoxic or normoxia conditions were set to 1.0. Values were expressed as the mean ± S.D. (error bars) of three different plates. N, Normoxia; ctrl, control; ##, non-specific band. * p<0.05; ** p<0.01, significantly different from mock cells in the presence of dipyridyl or DMOG or si-ctrl cells.