Figure 2.

Transcriptional activity as well as protein stability of HIF-1α is enhanced in the presence of MTA1. (A) The HRE-tk-Luc (0.1 μg) or VEGF promoter (−2003∼23)-Luc (0.3 μg) reporter was cotransfected with the indicated amount of eucaryotic expression vector for MTA1 into MCF-7 cells. Transfected cells were incubated for 24 h in the presence or absence of 100 μM CoCl₂. Luciferase activity was measured and normalized by β-gal activity. (B) MCF-7 or MDA-MB-231 cells were transfected with 3 μg of each pCMV-Myc-MTA1 or empty vector. Transfected cells were incubated under hypoxic stress, 0.1% O₂ or 100 μM CoCl₂, or normoxia for 24 h as indicated. The expression of HIF-1α, MTA1, Myc-MTA1, and VEGF was analyzed by Western blot (WB) analysis and RT–PCR. The expression of α-tubulin or β-actin was analyzed for control. (C) MDA-MB-231 cells were transfected twice at 24 h intervals with 200 nM of each control nonspecific siRNA (si-GL3) or human MTA1 siRNA (si-MTA1#1 or si-MTA1#2). After 3 h of the last transfection, the cells were incubated under hypoxic stress, 0.1% O₂ or 100 μM CoCl₂, or normoxia for 24 h as indicated. The expression of HIF-1α and MTA1 was analyzed by WB and RT–PCR. The expression of α-tubulin or β-actin was analyzed for control. (D) MDA-MB-231 cells were transfected with 3 μg of each pCMV-Myc-MTA1 (▪) or empty vector. The cells transfected with empty vector were incubated under hypoxia (•) or normoxia (▴) for 24 h as indicated. At the end of treatment, 10 μM cycloheximide (CHX) was added into the media for the indicated time periods. The expression of HIF-1α, Myc-MTA1, and α-tubulin was analyzed by WB analysis. The density of HIF-1α protein band was determined using an image analysis system. The values were normalized to that of α-tubulin and expressed as percent of the CHX-untreated control. (E) HEK293 cells that were plated the previous day on four-chamber slide glass were transfected with 0.5 μg of each Red-HIF-1α and/or GFP-MTA1. Transfected cells were treated with or without 100 μM CoCl₂ for 24 h. Cells were fixed and visualized by immunofluorescence microscopy. DAPI was used to stain nuclei. (F) MCF-7 cells were transfected with 3 μg of each pCMV-Myc-MTA1 or empty vector. Transfected cells were incubated under hypoxia or normoxia for 24 h as indicated. Cytoplasmic and nuclear extracts were obtained and the expression of HIF-1α, MTA1, Myc-MTA1, and α-tubulin was analyzed by WB analysis.