![Figure 7. Induction of HIF-1α transactivation by BAPTA-am via inhibition of prolyl hydroxylase activity. (A) HepG2 cells were cotransfected with a luciferase reporter construct pG5-E1B-LUC and different fusion gene constructs in which the Gal4 DNA binding domain was fused to either the HIF-1α region from amino acid 532 to 585 containing TADN or 727 to 826 containing TADC, respectively, as shown on the left side of panel A. After 24 hours the transfected cells were treated with either 5 μM BAPTA-am or 0.1 μM A23187 under normoxia (16% O2) or hypoxia (8% O2) for 24 hours. Values are means ± SEM of 6 independent culture experiments. Statistics, Student t test for paired values: *P ≤ .05 versus control in the same group. (B) Western blot analysis. Protein (50 μg) from HepG2 cells transfected with wild-type and mutated Gal4-HIF1αTADN and Gal4-HIF1αTADC constructs was treated with BAPTA-am (5 μM) or BAPTA (5 μM) for 4 hours and then subjected to Western analysis with an antibody against Gal4DBD. Autoradiographic signals were obtained by chemiluminescence. (C) In vitro prolyl hydroxylase activity assay. The GST-HIF1α-TADN fusion protein or the GST protein was incubated with HepG2 cell extract, cofactors, and [5-14C]2-oxoglutarate in the presence of CoCl2 (10 μM) or BAPTA (5 μM). The radioactivity associated to 14C-succinate was determined. In each experiment the basal HIF-TADN-dependent activity (control) was set to 100% after normalized by subtracting the GST-associated activity. Values are means ± SEM of 3 independent culture experiments. Statistics, Student t test for paired values: *P ≤ .05 versus control. (D) GST pull-down assay. HepG2 cells were treated with or without BAPTA-am (5 μM). Cell extracts were prepared and incubated with the GST-HIF1α-TADN fusion protein in the presence of CoCl2 (10 μM) or BAPTA (5 μM) supplemented with cofactors. Glutathione-Sepharose beads and [35S]VHL were then added, and the bound VHL was recovered, subjected to SDS-PAGE, and visualized by phosphoimaging. The input remains from directly loaded [35S]VHL. The 2 bands represent the 213- and 160-amino acid VHL translation products. Representative data of 3 individual experiments. (E) GST pull-down assay. Cell extracts were incubated as in panel D with the GST-p300CH1 protein and [35S]HIF-1α, and the bound HIF-1α was recovered, subjected to SDS-PAGE, and visualized by phosphoimaging. The input remains from directly loaded [35S]HIF-1α.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/104/13/10.1182_blood-2004-03-1017/6/zh80240470700007.jpeg?Expires=1724262912&Signature=tUYfmWMtzeLSvYLYPs4-AD2EJ~zRQgQK1MBa2LpB8m-SyWfCFURoHWlHmnyOjGVWwee2mTE-dqlbsH~k7p6ZvVX9coUFkINzg~Fn4lEn9vdv4rGXuz4kW1NkG7ZtWE1bIHYourreTGDcsgGL7s-gU1ScbVdDciKkasBvoJY4vuJABKpHA1O02qCgnTyrrlyB-ZwfpbtcwTWz5AW3j0dYVhhpMzCOl--ZNxWqWcRBqWqx2QlVAXo~iM5oFnQz83MP3PQq~fNiA6ocwAs0YoA9m8Pjtq4Wv4NQ2d2fYv52QgQMGtP2EkDfyx6ZVpNb1TKbGAT~oSmK98feoth28l6aLQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Induction of HIF-1α transactivation by BAPTA-am via inhibition of prolyl hydroxylase activity. (A) HepG2 cells were cotransfected with a luciferase reporter construct pG5-E1B-LUC and different fusion gene constructs in which the Gal4 DNA binding domain was fused to either the HIF-1α region from amino acid 532 to 585 containing TADN or 727 to 826 containing TADC, respectively, as shown on the left side of panel A. After 24 hours the transfected cells were treated with either 5 μM BAPTA-am or 0.1 μM A23187 under normoxia (16% O2) or hypoxia (8% O2) for 24 hours. Values are means ± SEM of 6 independent culture experiments. Statistics, Student t test for paired values: *P ≤ .05 versus control in the same group. (B) Western blot analysis. Protein (50 μg) from HepG2 cells transfected with wild-type and mutated Gal4-HIF1αTADN and Gal4-HIF1αTADC constructs was treated with BAPTA-am (5 μM) or BAPTA (5 μM) for 4 hours and then subjected to Western analysis with an antibody against Gal4DBD. Autoradiographic signals were obtained by chemiluminescence. (C) In vitro prolyl hydroxylase activity assay. The GST-HIF1α-TADN fusion protein or the GST protein was incubated with HepG2 cell extract, cofactors, and [5-14C]2-oxoglutarate in the presence of CoCl2 (10 μM) or BAPTA (5 μM). The radioactivity associated to 14C-succinate was determined. In each experiment the basal HIF-TADN-dependent activity (control) was set to 100% after normalized by subtracting the GST-associated activity. Values are means ± SEM of 3 independent culture experiments. Statistics, Student t test for paired values: *P ≤ .05 versus control. (D) GST pull-down assay. HepG2 cells were treated with or without BAPTA-am (5 μM). Cell extracts were prepared and incubated with the GST-HIF1α-TADN fusion protein in the presence of CoCl2 (10 μM) or BAPTA (5 μM) supplemented with cofactors. Glutathione-Sepharose beads and [35S]VHL were then added, and the bound VHL was recovered, subjected to SDS-PAGE, and visualized by phosphoimaging. The input remains from directly loaded [35S]VHL. The 2 bands represent the 213- and 160-amino acid VHL translation products. Representative data of 3 individual experiments. (E) GST pull-down assay. Cell extracts were incubated as in panel D with the GST-p300CH1 protein and [35S]HIF-1α, and the bound HIF-1α was recovered, subjected to SDS-PAGE, and visualized by phosphoimaging. The input remains from directly loaded [35S]HIF-1α.
