Interaction of Nkx2.5 and <i>O-</i>GlcNAc transferase (OGT).

<p>(A) Immunoblotting analysis of myc-Nkx2.5 and <i>O</i>-GlycNAcylated proteins in myc-Nkx2.5 and OGT-flag-co-transfected HEK293 cells. (B) Graphs showed relative <i>O</i>-GlcNAc (left) and myc-Nkx2.5 (right) levels normalized to the tubulin levels in co-transfected cells and single transfected cells. <i>O</i>-GlcNAcylation levels were compared in no-transfected cells <i>vs</i> OGT-flag cells and myc-Nkx2.5 cells <i>vs</i> OGT-flag/myc-Nkx2.5 cells. Myc-Nkx2.5 protein levels were compared in myc-Nkx2.5 cells <i>vs</i> OGT-flag/myc-Nkx2.5 cells. All values are presented as the mean±standard error of three independent experiments. *<i>p</i><0.05. (C) After immunoprecipitation with antibodies specific for Nkx2.5 and flag, immunoblotting with antibodies for myc, flag, and OGT was performed. (D) After immunoprecipitation with antibodies specific for OGT, immunoblotting with antibodies for myc, flag, and OGT was performed. The interaction between myc-Nkx2.5 and OGT-flag was observed.</p

Expression of Nkx2.5 in the heart tissue of diabetic mice (DM).

<p>(A) Immunoblotting analysis of Nkx2.5 and <i>O</i>-GlycNAcylated proteins in the heart tissue of DM intraperitoneally injected with streptozotocine (180 mg/kg body weight). (B) Graphs showed relative <i>O</i>-GlcNAc (left) and Nkx2.5 (right) levels normalized to the tubulin levels in DM and control. Nkx2.5 protein in diabetic heart of mice was significantly decreased. <i>O</i>-GlcNAcylation and Nkx2.5 protein levels were compared in DM <i>vs</i> control. All values are presented as the mean±standard error of three independent experiments. *<i>p</i><0.05 DM vs. control.</p

Reduction of myc-Nkx2.5 protein in cells treated with <i>O</i>-GlcNAcase inhibotirs.

<p>(A) Immunoblotting of myc-Nkx2.5 and <i>O</i>-GlycNAcylated proteins in myc-Nkx2.5-transfected HEK293 cells in the presence or absence of 3 mM streptozotocine (STZ). (B) Graphs showed relative <i>O</i>-GlcNAc (left) and myc-Nkx2.5 (right) levels normalized to the tubulin levels in STZ-treated and untreated cells. <i>O</i>-GlcNAcylation levels were compared in untreated cells <i>vs</i> STZ-treated cells and untreated myc-Nkx2.5 cells <i>vs</i> STZ-treated myc-Nkx2.5 cells. The myc-Nkx2.5 protein levels were compared in untreated myc-Nkx2.5 cells <i>vs</i> STZ-treated myc-Nkx2.5 cells. All values are presented as the mean±standard error of three independent experiments. *<i>p</i><0.05. (C) Immunoblotting analysis of myc-Nkx2.5 and <i>O</i>-GlycNAcylated proteins in myc-Nkx2.5-transduced HEK293 cells in the presence or absence of <i>O</i>-(2-acetamido-2-deoxy-D-glucopyroanosylidene)-amino-<i>N</i>-phenylcarbamate (10 or 100 µM PUGNAC). (D) Graphs showed relative <i>O</i>-GlcNAc (left) and myc-Nkx2.5 (right) levels normalized to the tubulin levels in PUGNAC-treated and untreated cells. The treatment with STZ and PUGNAC significantly increased the <i>O</i>-GlcNAcylation of intracellular proteins, but decreased the myc-Nkx2.5 protein significantly. <i>O</i>-GlcNAcylation levels were compared in untreated myc-Nkx2.5 cells <i>vs</i> PUGNAC-treated myc-Nkx2.5 cells. The myc-Nkx2.5 protein levels were compared in untreated myc-Nkx2.5 cells <i>vs</i> PUGNAC treated myc-Nkx2.5 cells. All values are presented as the mean±standard error of three independent experiments. *<i>p</i><0.05.</p

Modification of Nkx2.5 by <i>O</i>-GlcNAc.

<p>(A) The lysates of myc-Nkx2.5 and OGT-flag-co-transfected HEK293 cells were subjected to immunoprecipitation with anti-myc antibody and immunoblottings were performed with antibodies against <i>O</i>-GlcNAc (CTD110.6, RL-2) and myc. The modification of myc-Nkx2.5 with O-GlcNAc was detected. (B) Diabetic mice (DM) maintained elevated blood glucose level (>430 mg/dL) at 3 and 7 days after injection with streptozotocine (180 mg/kg body weight). The heart homogenates of control and diabetic mice were immunoprecipitated with anti-Nkx2.5 antibody, followed by immunoblottings with RL-2 antibodies against <i>O</i>-GlcNAc and anti-Nkx2.5 antibody. Nkx2.5 proteins of heart tissues were modified with <i>O</i>-GlcNAc and this modification increased in diabetic mice compared with control mice.</p

Facile Preparation of Zwitterion-Stabilized Superparamagnetic Iron Oxide Nanoparticles (ZSPIONs) as an MR Contrast Agent for in Vivo Applications

We describe a simple method for synthesizing superparamagnetic nanoparticles (SPIONs) as small, stable contrast agents for magnetic resonance imaging (MRI) based on sulfobetaine zwitterionic ligands. SPIONs synthesized by thermal decomposition were coated with zwitterions to impart water dispersibility and high in vivo stability through the nanoemulsion method. Zwitterion surfactant coating layers are formed easily on oleic acid-stabilized SPIONs via hydrophobic and van der Waals interactions. Our zwitterion-coated SPIONs (ZSPIONs) had ultrathin (∼5 nm) coating layers with mean sizes of 12.0 ± 2.5 nm, as measured by dynamic light scattering (DLS). Upon incubation in 1 M NaCl and 10% FBS, the ZSPIONs showed high colloidal stabilities without precipitating, as monitored by DLS. The T2 relaxivity coefficient of the ZSPIONs, obtained by measuring the relaxation rate on the basis of the iron concentration, was 261 mM^–1s^–1. This value was much higher than that of the commercial T2 contrast agent because of the ultrathin coating layer. Furthermore, we confirmed that ZSPIONs can be used as MR contrast agents for in vivo applications such as tumor imaging and lymph node mapping

Ferritin-based MRI of dendritic cell

Figure 1. Analysis of dendritic cell (DC) transduced with myc-tagged human ferritin heavy chain (FTH) and green fluorescence prtein (GFP) using lentivirus. Figure 2. Analyses of proliferation and migration activities and co-stimulatory molecules expressions in dendritic cell (DC) and human ferritin heavy chain-transduced DC (FTH-DC). Figure. 3. Cellular iron staining, iron amount measurement and in vitro MRI analysis of dendritic cell (DC) and human ferritin heavy chain-transduced DC (FTH-DC). Figure 4. in vivo and ex vivo MRI of popliteal lymph nodes (LNs) of mouse injected with dendritic cell (DC) and ferritin heavy chain-transduced DC (FTH-DC). Figure 5. Histological analysis of cryosectioned popliteal lymph nodes (LNs) with dendritic cell (DC) and ferritin heavy chain-transduced DC (FTH-DC)

Histological analysis of cryosectioned popliteal lymph nodes (LNs) with dendritic cell (DC) and ferritin heavy chain-transduced DC (FTH-DC).

<p>(A) Representative GFP fluorescence images of popliteal LNs isolated from mouse at 48 h after injection of DCs and FTH-DCs. (B) Representative hematoxylin and eosin staining of cryosectioned popliteal LNs with DCs and FTH-DCs. (C) Immunofluorescence images of GFP (green) and FTH (red) detected with anti-GFP and anti-myc antibodies in LNs with DCs and FTH-DCs. (D) Merge images (yellow) of co-immunofluorescence staining of activation marker, CD25 (red) and GFP (green) detected with anti-CD25 and anti-GFP antibodies in FTH-DCs of cryosectioned LNs. Nuclear was stained with diamidino-2-phenylindole (DAPI, blue).</p

Analyses of proliferation and migration activities and co-stimulatory molecules expressions in dendritic cell (DC) and human ferritin heavy chain-transduced DC (FTH-DC).

<p>(A) A standard 3-,5-diphenyltetrazolium bromide (MTT) assay for proliferation activity of DCs and FTH-DCs cultured for 24 h, 48 h and 72 h. (B and C) Trans-well assay for migration abilities of DCs and FTH-DCs incubated with TNF-α (20 ng/mL) and IFN-γ (20 ng/mL) in the presence or absence of CCL19 and CCL21 for 24 h. Representative fluorescent image of nuclear stained with diamidino-2-phenylindole (DAPI) in DCs and FTH-DCs that migrated to the lower chamber. (D) RT-PCR analysis of C-C chemokine receptor type-7 (CCR-7) in DCs and FTH-DCs. Both DCs and FTH-DCs, which were incubated in the medium supplemented with TNF-α (20 ng/mL) and IFN-γ (20 ng/mL) for 24 h, highly expressed the CCR-7 as compared to untreated cells. (E and F) Representative flow cytometric analysis of co-stimulatory molecules such as CD40, CD80 and CD86 in DCs and FTH-DCs treated with or without LPS (100 ng/mL) for 24 h. Flow cytometric results obtained from 3 independent experiments. All data are presented as the mean ± standard deviations of at least three independent experiments. *, <i>p</i> ≤0.05.</p

<i>in vivo</i> and <i>ex vivo</i> MRI of popliteal lymph nodes (LNs) of mouse injected with dendritic cell (DC) and ferritin heavy chain-transduced DC (FTH-DC).

<p>(A) Representative <i>in vivo</i> T₂*-weighted images of popliteal LNs (circle) of mouse before and at 48 h after injection of 1x10⁷ DCs and FTH-DCs. (B) Average R₂* values measured from <i>in vivo</i> popliteal LNs with DCs and FTH-DCs. (C) Representative <i>ex vivo</i> T₂*-weighted images of popliteal LNs isolated from mouse at 48 h after injection of DCs and FTH-DCs. (D) Average R₂* values measured from <i>ex vivo</i> popliteal LNs with DCs and FTH-DCs. All data are presented as the mean ± standard deviations of at least three independent experiments. *, <i>p</i>≤0.05</p

Cellular iron staining, iron amount measurement and in vitro MRI analysis of dendritic cell (DC) and human ferritin heavy chain-transduced DC (FTH-DC).

<p>(A) Representative prussian blue staining of cellular iron in DCs and FTH-DCs incubated with or without 250 μM ferric ammonium citrate (FAC) for 24 h. (B) Average cellular iron amount measured from DCs and FTH-DCs incubated with 25 μM and 250 μM FAC. (C) Representative T₂*-weighted images and color-coded of the R₂* values in the DCs and FTH-DCs phantoms. (D) Average R₂* values measured from DCs and FTH-DCs phantoms. All data are presented as the mean ± standard deviations of at least three independent experiments. *, <i>p</i> ≤0.05.</p