The role of HIF-1α in hypoxia-induced NMBR expression.

<p>(A) MDA-MB-231 cells were exposed to hypoxia induced using 400 or 800 μM l-mimosine for 16 h. Western blot analysis was performed using antibodies specific for human NMB-R and HIF-1α, and α-tubulin served as a loading control (left). The right panel shows the densitometric analysis assessing relative NMB-R and HIF-1α expression levels. *P<0.05 vs. control. B and C, MDA-MB-231 cells were exposed to 0.5 mM DMOG under normoxic conditions for the indicated times. (B) RT-PCR analysis was performed using specific primers for NMB-R or β-actin. The expression of NMB-R was normalized to that of the internal control β-actin (left). The density of the control bands (untreated) was defined as 100% (right). *P<0.05 vs. control. (C) Western blots were probed with anti-NMB-R or anti-HIF-1α antibodies, and α-tubulin served as a loading control (left). The right panel shows the densitometric analysis of relative NMB-R and HIF-1α expression levels. *P<0.05 vs. control. (D) MDA-MB-231 cells were transfected with an HIF-1α expression vector and then exposed to hypoxic or normoxic conditions. Western blot analysis of cell lysates using anti-NMB-R or anti-HIF-1α antibodies was performed, and α-tubulin served as a loading control (left). The graph shows the densitometric analysis of the relative NMB-R levels (right). The results represent at least 3 independent experiments. *P<0.05 vs. control empty vector. </p

Localization of HREs in the NMBR promoter.

<p>(A) The location of putative HRE sites and the most conserved elements within the ~1 kb fragment of the 5'-flanking region of human NMBR. Putative HRE sites are defined by the core sequence 5'-RCGTG-3'. The 1259-bp fragment of the 5'-flanking region of human NMBR were subcloned upstream of a luciferase reporter gene. (B) MDA-MB-231 cells were transiently transfected with each of the NMBR reporter vectors and pCMV-β-galactosidase and then incubated under normoxic or hypoxic conditions for 24 h. The cell extracts were analyzed for luciferase activity. *P<0.05 and **P<0.01 vs. pGL3 alone. (C) MDA-MB-231 cells were cotransfected with p(1259)luc, pBOS-hHIF-1α, and pBOS-hARNT and then incubated under normoxic or hypoxic conditions for 24 h. Luciferase activity was determined. *P<0.05 and **P<0.01 vs. mock. (D) MDA-MB-231 cells were cotransfected with p(1259)luc and an HIF-1α or HIF-2α expression vector as indicated and then incubated under hypoxic conditions for 24 h. Luciferase activity was determined. Data represent 3 independent experiments. *P<0.05 vs. pGL3.</p

Effect of hypoxia on NMBR expression.

<p>MDA-MB-231 cells were incubated under normoxic (21% O₂) or hypoxic (1% O₂) conditions for the indicated times. (A) Total RNA was isolated and analyzed using RT-PCR with primers specific for human NMBR. β-actin was used as the internal control. (B) Using real-time PCR, the expression levels of NMBR mRNA were quantified. The expression level of the control (untreated) was defined as 100%, and the values were normalized to those of β-actin mRNA levels. (C) The expression of NMB-R was examined by western blotting using an anti-NMB-R antibody, and α-tubulin served as the loading control (left). The right panel shows the densitometric analysis of relative NMB-R expression levels in at least 3 independent experiments. *P<0.05 vs. control. (D) Higher magnification images showed immunoreactivity of NMB-R (red) and HIF-1α (green) in MDA-MB-231 cells under hypoxic or normoxic conditions. </p

Effect of HIF-1α knockdown on hypoxia-induced NMBR expression.

<p>MDA-MB-231 cells were treated with 100 μM YC-1 under normoxic or hypoxic conditions for 8 h. (A) RT-PCR analysis was performed using specific primers for human NMB-R. β-actin served as an internal control. The graph shows the densitometric analysis of the relative NMBR mRNA levels (right). **P<0.01 vs. normoxia; ^#P<0.05 vs. hypoxia. (B) Western blot analysis was performed using antibodies specific for human NMB-R and HIF-1α, and α-tubulin served as a loading control (left). The right panel shows the densitometric analysis of relative NMB-R and HIF-1α expression levels. *P<0.05 vs. normoxia; ^#P<0.05 vs. hypoxia. . (C) MDA-MB-231 cells were transiently transfected with HIF-1α siRNA or control siRNAs. After transfection, the cells were incubated under normoxic or hypoxic conditions and subjected to western blot analysis to detect NMB-R or HIF-1α (left). The relative NMB-R expression level was measured in at least 3 independent experiments. *P<0.05 and **P<0.01 vs. normoxia; ^#P<0.05 vs. control siRNA.</p

The role of HIF-1α in hypoxia-induced NMBR expression.

<p>(A) MDA-MB-231 cells were exposed to hypoxia induced using 400 or 800 μM l-mimosine for 16 h. Western blot analysis was performed using antibodies specific for human NMB-R and HIF-1α, and α-tubulin served as a loading control (left). The right panel shows the densitometric analysis assessing relative NMB-R and HIF-1α expression levels. *P<0.05 vs. control. B and C, MDA-MB-231 cells were exposed to 0.5 mM DMOG under normoxic conditions for the indicated times. (B) RT-PCR analysis was performed using specific primers for NMB-R or β-actin. The expression of NMB-R was normalized to that of the internal control β-actin (left). The density of the control bands (untreated) was defined as 100% (right). *P<0.05 vs. control. (C) Western blots were probed with anti-NMB-R or anti-HIF-1α antibodies, and α-tubulin served as a loading control (left). The right panel shows the densitometric analysis of relative NMB-R and HIF-1α expression levels. *P<0.05 vs. control. (D) MDA-MB-231 cells were transfected with an HIF-1α expression vector and then exposed to hypoxic or normoxic conditions. Western blot analysis of cell lysates using anti-NMB-R or anti-HIF-1α antibodies was performed, and α-tubulin served as a loading control (left). The graph shows the densitometric analysis of the relative NMB-R levels (right). The results represent at least 3 independent experiments. *P<0.05 vs. control empty vector. </p

Expression of NMB-R and HIF-1α in breast carcinoma.

<p>(A) MDA-MB-231 cells were injected in the flanks of nude mice. Prior to sacrifice, MDA-MB-231 xenografts were intravenously injected with Hypoxyprobe-1 (60 mg/kg) and then embedded using OCT compound. Tissue sections from tumor xenografts were analyzed using immunohistochemistry for expression of Hypoxyprobe-1 (green), NMB-R (red), and HIF-1α (red). (B) Tissue microarray slides for human breast cancers were double-immunostained with anti-NMB-R (red) and anti-HIF-1α (green) antibodies, respectively. Sections were stained without primary anti-NMB-R or anti-HIF-1α antibodies as controls. Representative tumor sections are shown. (C) The table presents the frequency of NMB-R and HIF-1α expression in infiltrating, metastatic, sarcomatoid, intraductal papillary, and atypical medullary breast carcinoma tissues.</p