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

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

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

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

DataSheet1_Lithium doped biphasic calcium phosphate: Structural analysis and osteo/odontogenic potential in vitro.PDF

Biphasic calcium phosphate (BCP) is generally considered a good synthetic bone graft material with osteoinductive potential. Lithium ions are trace elements that play a role in the bone-remodeling process. This study aimed to investigate the effects of lithium ions on the phase, crystal structure, and biological responses of lithium doped BCPs and to identify improvements in their osteogenic properties. Lithium-doped BCP powders with different doping levels (0, 5, 10, and 20 at%) were synthesized via the co-precipitation method. We found that the four types of lithium-doped BCP powders showed different phase compositions of hydroxyapatite and β-tricalcium phosphate. In addition, lithium ions favored entering the β-tricalcium phosphate structure at the Ca (4) sites and calcium vacancy sites [VCa(4)] up to 10 at%. This substitution improves the crystal stabilization by filling the vacancies with Ca2+ and Li+ in all Ca sites. However, when the concentration of Li ions was higher than 10 at%, lithium-induced crystal instability resulted in the burst release of lithium ions, and the osteogenic behavior of human dental pulp stem cells did not improve further. Although lithium ions regulate osteogenic properties, it is important to determine the optimal amount of lithium in BCPs. In this study, the most effective lithium doping level in BCP was approximately 10 at% to improve its biological properties and facilitate medical applications.</p