Expression of MTA1 is induced under hypoxia. (A) MCF-7 or MDA-MB-231 cells were incubated in hypoxia for the indicated time periods. The expression of HIF-1α and MTA1 was analyzed by Western blot (WB) analysis and RT–PCR. The expression of α-tubulin or β-actin was analyzed for control. (B) T47D, MCF-7, MDA-MB-231, and MDA-MB-435 cells were incubated under hypoxia or normoxia for 24 h as indicated. The expression of MTA1, HIF-1α, and α-tubulin was analyzed by WB analysis. (C) MCF-7 cells were transfected with 4 μg of each pEBG-HIF-1α or empty vector. The expression of MTA1, GST-HIF-1α, GST, and α-tubulin was analyzed by WB analysis. (D) MCF-7 or MDA-MB-231 cells were transfected twice at 24 h intervals with 200 nM of each control nonspecific siRNA, si-GL3, or human HIF-1α siRNA, si-HIF-1α. After 3 h of the last transfection, the cells were incubated under hypoxia or normoxia for 24 h. The expression of MTA1, HIF-1α, and α-tubulin was analyzed by WB analysis.

MTA1 interacts with HIF-1α in vivo. (A) MCF-7 cells were incubated in the presence of 100 μM CoCl₂ for 24 h. Whole-cell lysates (500 μg) were immunoprecipitated (IP) with normal mouse IgG, anti-HIF-1α, anti-MTA antibodies, and then probed using anti-HIF-1α and anti-MTA1 antibodies. The expression of HIF-1α, MTA1, and α-tubulin was analyzed by Western blot (WB) analysis. (B) Schematic representation of full-length and deletion HIF-1α constructs containing the basic helix–loop-helix (bHLH)/PER–ARNT–SIM (PAS) (N1 and N2), ODD domain, inhibitory domain (ID), N-terminal transactivation domain (NTAD), and C-terminal transactivation domain (CTAD). Nuclear localization signal (NLS) was also indicated. (C) MCF-7 cells were transfected with 3 μg of each pEBG-HIF-1α deletion construct and pCMV-Myc-MTA1. Whole-cell lysates (500 μg) were IP with anti-GST antibody, and then probed using anti-Myc antibody. The expression of GST-HIF-1α deletions, Myc-MTA1, and α-tubulin was analyzed by WB analysis.

ODD domain of HIF-1α is deacetylated in the presence of MTA1. (A) MCF-7 cells were transfected with 3 μg each of the pEBG-HIF-1α deletion construct (Figure 3B), pCMV-Myc-MTA1, or empty vector. After 24 h of transfection, 500 μg of whole-cell lysates were immunoprecipitated (IP) with anti-pan-Ac antibody, and then probed using anti-GST antibody. The expression of GST-HIF-1α deletions, Myc-MTA1, and α-tubulin was analyzed by Western blot (WB) analysis. (B) MCF-7 cells were transfected with 3 μg each of pEBG-HIF-1α ODD (GST-ODD), pCMV-Myc-MTA1 (•), or empty vector. After 1 h of transfection, the cells transfected with empty vector were treated with (▪) or without (▴) 100 μM CoCl₂ for 24 h. At the end of treatment, 10 μM cycloheximide (CHX) was added into the media for the indicated time periods. The expression of GST-ODD, Myc-MTA1, and α-tubulin was analyzed by WB analysis. The density of GST-ODD 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.

MTA1 interferes the association of HIF-1α and VHL and PHDs. (A) 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. Cells were treated with or without 10 μM MG132 for 1 h before being harvested. Whole-cell lysates (500 μg) were immunoprecipitated (IP) with anti-HIF-1α antibody, and then probed using anti-VHL antibody. The expression of HIF-1α, VHL, Myc-MTA1, and α-tubulin was analyzed by Western blot (WB) analysis. (B) MCF-7 cells were transfected with 3 μg of each pEGFP-N1-PHD1 (GFP-PHD1), pEGFP-N1-PHD2 (GFP-PHD2), or pEGFP-N1-PHD3 (GFP-PHD3), with or without 3 μg pCMV-Myc-MTA1 as indicated. Transfected cells were incubated under hypoxia or normoxia for 24 h. Cells were treated with or without 10 μM MG132 for 1 h before being harvested. Whole-cell lysates (500 μg) were IP with anti-pan-Ac and anti-HIF-1α antibodies, and then probed using anti-HIF-1α or anti-GFP antibodies. The expression of HIF-1α, GFP-PHDs, Myc-MTA1, and α-tubulin was analyzed by WB analysis. (C) MCF-7 cells were transfected with 3 μg of each pEBG-HIF-1α ODD (GST-ODD), pCMV-Myc-VHL, and pEGFP-N1-PHD2 (GFP-PHD2). For Myc-MTA1, transfection was repeated with 3 μg of each pCMV-Myc-MTA1 or empty vector after 24 h of the first transfection. After incubating for 24 h, the cells were exposed to hypoxia or normoxia for 24 h. For si-MTA1 transfection, 200 nM human MTA1 siRNA (si-MTA1) was transfected after 24 h of the first transfection, and repeated once after 21 h. After 3 h of the last transfection, the cells were incubated under hypoxia or normoxia for 24 h. Cells were treated with or without 10 μM MG132 for 1 h before being harvested. Whole-cell lysates (500 μg) were IP with anti-GST, or anti-pan-Ac antibodies, and then probed using anti-Myc, anti-GFP, or anti-GST antibodies. The expression of GST-ODD, Myc-VHL, GFP-PHD2, MTA1, and α-tubulin was analyzed by WB analysis.

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.

HDAC1 is involved in the MTA1-induced stabilization of HIF-1α. (A) MCF-7 cells were transfected with 3 μg of each pCMV-Myc-MTA1 or empty vector. Transfected cells were treated with or without 100 μM CoCl₂ for 24 h as indicated. Cells were treated with or without 300 ng/ml TSA for 3 h as indicated before being harvested. The expression of HIF-1α, HDAC1, MTA1, Myc-MTA1, and α-tubulin was analyzed by Western blot (WB) analysis. (B) 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. Cells were treated with 10 μM MG132 or 300 ng/ml TSA for 1 h or 3 h, respectively, before cells were harvested. Whole-cell lysates (500 μg) were immunoprecipitated (IP) with anti-pan-Ac antibody, and then probed using anti-HIF-1α antibody. The expression of HIF-1α, HDAC1, MTA1, Myc-MTA1, and α-tubulin was analyzed by WB analysis. (C) MCF-7 cells were incubated in the presence of 100 μM CoCl₂ for 24 h. Whole-cell lysates (500 μg) were IP with normal mouse IgG, anti-HIF-1α, anti-MTA1 antibodies, and then probed using anti-HDAC1 antibody. The expression of HIF-1α, HDAC1, Myc-MTA1, and α-tubulin was analyzed by WB analysis.

MTA1 counteracts to the ARD1-induced degradation of HIF-1α. (A) HEK293, HeLa, HepG2, and MCF-7 cells were treated with or without 100 μM CoCl₂ for 24 h. The expression of HIF-1α, MTA1, and α-tubulin was analyzed by Western blot (WB) analysis. (B) HEK293, HeLa, HepG2, and MCF-7 cells were transfected with 3 μg of pCMV-Tag2C-FLAG-mARD1²²⁵ (F-ARD1) or empty vector. Transfected cells were incubated under hypoxia or normoxia for 24 h. The expression of HIF-1α, MTA1, FLAG-ARD1 (F-ARD1), and α-tubulin was analyzed by WB analysis. (C) HEK293 cells were transfected with 9 μg of pCMV-Tag2C-FLAG-mARD1²²⁵ (FLAG-ARD1) or the indicated amount of pCMV-Myc-MTA1. The expression of HIF-1α, Myc-MTA1, FLAG-ARD1, and α-tubulin was analyzed by WB analysis. (D) MCF-7 cells were transfected with 4 μg pEBG-HIF-1α (GST-HIF-1α), and 3 μg of each pCMV-Tag2C-FLAG-mARD1²²⁵ (FLAG-ARD1), pCMV-Myc-MTA1, or empty vector. Cells were treated with or without 10 μM MG132 for 1 h before being harvested. Whole-cell lysates (500 μg) were immunoprecipitated (IP) with anti-pan-Ac antibody, and then probed using anti-GST antibody. The expression of GST-HIF-1α, FLAG-ARD1, Myc-MTA1, and α-tubulin was analyzed by WB analysis. (E) MCF-7 cells were transfected with 4 μg pEBG-HIF-1α (GST-HIF-1α) with 3 μg of each pCMV-Tag2C-FLAG-mARD1²²⁵ or pCMV-Myc-MTA1 as indicated. After 1 h of transfection, cells were incubated under hypoxia or normoxia for 24 h as indicated. Cells were treated with or without 300 ng/ml TSA for 3 h before being harvested. The expression of GST-HIF-1α, FLAG-ARD1, Myc-MTA1, and α-tubulin was analyzed by WB analysis. (F) HEK293 cells were transfected with the indicated combinations of 1 μg of each p3XFLAG^TM7.1-HIF-1α (WT), or p3XFLAG^TM7.1-HIF-1α K532R (K532R), 3 μg of each Myc-MTA1 and empty vector. After 24 h of transfection, cells were incubated in the presence or absence of 300 ng/ml TSA for 3 h before being harvested. The expression of FLAG-HIF-1α, Myc-MTA1, and α-tubulin was analyzed by WB analysis.