Expression of Le^y in Oral Cells.

<p>(<b>A</b>) Analysis of FUT1, FUT2, and FUT4 mRNA expression in four oral cell lines as determined using real-time PCR. The relative abundance of transcripts was obtained by normalization to GAPDH. Data represent the mean ± SEM (n = 3). The relative abundance shown in the y-axis was 100 times the results. n.s.: not significant, #<i>P</i> < 0.05 and &<i>P</i> < 0.001 compared to HSC-3. *<i>P</i> < 0.05, *<i>P</i> < 0.01 and ***<i>P</i> < 0.001. (<b>B</b>) Representative Western blot of Le^y and α-tubulin in four oral cell lines.</p

Le^y Suppression Accelerates Ligand-Induced Degradation of EGFR.

<p>After 24 h of serum-starvation, cells were treated with 40 ng/mL EGF for the indicated durations, and total cell lysates (<b>A</b>) or cell membrane fractions (<b>B</b>) were used for Western blotting using anti-EGFR antibody.</p

Le^y Glycosylation of EGFR in OC-2 Cells.

<p>(<b>A</b>) Representative Western blot of EGFR and α-tubulin in four oral cell lines. (<b>B</b>) Total cell lysates were used for immunoprecipitation (IP) with anti-EGFR and isotype control antibodies and immunoblotted (IB) using anti-Le^y and anti-EGFR antibodies. (<b>C</b>) Representative Western blot of Le^y and α-tubulin in OC-2 cells transduced with FUT1 (shFUT1) and control shRNA (shLuc). (<b>D</b>) Total cell lysates prepared from OC-2 cells with FUT1 (shFUT1) and control shRNA (shLuc) transduction were used for immunoprecipitation (IP) with anti-EGFR and isotype control antibodies, followed by immunoblotting (IB) with anti-Le^y and anti-EGFR antibodies.</p

Le^y Suppression Attenuates EGF-induced Phosphorylation and Migration in OC-2 Cells.

<p>Cells were stimulated with EGF for the indicated durations (<b>A</b>) or with various doses of EGF for 1.5 min (<b>B</b>). Total cell lysates were used for Western blotting using the indicated antibodies. Western blots from three independent experiments were analyzed by densitometry. The indicated fold changes represent the density relative to control (-). (<b>C</b>) After preincubation of AG1478 (EGFR inhibitor) for 30 min, each cells was wounded and stimulated with EGF. The progress of cell migration was recorded by using time-lapse microscopy, and the area of wound closure was calculated. Representative images demonstrating wound closure with EGF and EGFR inhibitor treatment as indicated. Quantitative data are shown. Data are presented as the means ± SEM (n = 3). *<i>P</i> < 0.05, **<i>P</i> < 0.01, and ***<i>P</i> < 0.001. (<b>D</b>) OC-2 cells transduced with shLuc or shFUT1 were stimulated with EGF in the presence or absence of AG1478 (EGFR inhibitor), and cell growth was analyzed every 24 h using WST-1 reagent. Data represent the mean ± SEM (n = 3).</p

Effects of EGF Treatment in OC-2 Cells.

<p>(<b>A</b>) Cells were stimulated with EGF for the indicated duration, and total cell lysates were used for Western blotting using the indicated antibodies. (<b>B</b>) Cells were stimulated with (+) or without (-) EGF for 5 min in the presence of various doses of AG1478 (EGFR inhibitor), and total cell lysates were used for Western blotting using the indicated antibodies. (<b>C</b>) After preincubation of various dosages of AG1478 (EGFR inhibitor) for 30 min, cells were wounded and stimulated with EGF. The progress of cell migration was recorded by using time-lapse microscopy, and the percentage of wound closure was calculated. Representative images demonstrating wound closure with EGF and EGFR inhibitor treatment as indicated. Quantitative data are shown. Data are presented as the means ±SEM (n = 3). *<i>P</i> < 0.05 and ***<i>P</i> < 0.001 versus EGF only. #<i>P</i> < 0.001.</p

Lewis^y Promotes Migration of Oral Cancer Cells by Glycosylation of Epidermal Growth Factor Receptor

<div><p>Aberrant glycosylation changes normal cellular functions and represents a specific hallmark of cancer. Lewis^y (Le^y) carbohydrate upregulation has been reported in a variety of cancers, including oral squamous cell carcinoma (OSCC). A high level of Le^y expression is related to poor prognosis of patients with oral cancer. However, it is unclear how Le^y mediates oral cancer progression. In this study, the role of Le^y in OSCC was explored. Our data showed that Le^y was upregulated in HSC-3 and OC-2 OSCC cell lines. Particularly, glycosylation of epidermal growth factor receptor (EGFR) with Le^y was found in OC-2 cells, and this modification was absent upon inhibition of Le^y synthesis. The absence of Le^y glycosylation of EGFR weakened phosphorylation of AKT and ERK in response to epidermal growth factor (EGF). Additionally, EGF-triggered cell migration was reduced, but cell proliferation was not affected. Le^y modification stabilized EGFR upon ligand activation. Conversely, absence of Le^y glycosylation accelerated EGFR degradation. In summary, these results indicate that increased expression of Le^y in OSCC cells is able to promote cell migration by modifying EGFR which in turn stabilizes EGFR expression and downstream signaling. Targeting Le^y on EGFR could have a potential therapeutic effect on oral cancer.</p></div

Primers used in quantitative real-time PCR.

<p>Primers used in quantitative real-time PCR.</p

Additional file 1: of The chondroitin sulfate moiety mediates thrombomodulin-enhanced adhesion and migration of vascular smooth muscle cells

Supplementary data. (DOCX 315 kb

Increased TM expression in corneal epithelium injury.

<p>TM expression (green) at 6, 18, and 36 h after wounding was examined by immunofluorescence staining. Unwound (un) cornea was used as control. Arrows: TM expression in the unwounded cornea was relatively weaker compared with that in the wounded cornea. Nuclei were visualized by DAPI (blue) nuclear staining. Arrowheads: corneal epithelium; w: wounded area. Magnification: 400×. Bar = 25 μm.</p

The Time Window for Therapy with Peptide Nanofibers Combined with Autologous Bone Marrow Cells in Pigs after Acute Myocardial Infarction

<div><p>Background</p><p>We previously showed that injection of peptide nanofibers (NF) combined with autologous bone marrow mononuclear cells (MNC) immediately after coronary artery ligation improves cardiac performance in pigs. To evaluate the clinical feasibility, this study was performed to determine the therapeutic time window for NF/MNC therapy in acute myocardial infarction (MI).</p><p>Methods and Results</p><p>A total of 45 adult minipigs were randomly grouped into 7 groups: sham or MI plus treatment with NS (normal saline), or NF or MNC alone at 1 day (1D) post-MI, or NF/MNC at 1, 4, or 7 days post-MI (N≥6). Cardiac function was assessed by echocardiography and ventricular catheterization. Compared with the NS control, pigs treated with NF/MNC at 1 day post-MI (NF/MC-1D) had the greatest improvement in left ventricle ejection fraction (LVEF; 55.1±1.6%; P<0.01 vs. NS) 2 months after MI. In contrast, pigs treated with either NF/MNC-4D or NF/MNC-7D showed 48.9±0.8% (P<0.05 vs. NS) and 43.5±2.3% (n.s. vs. NS) improvements, respectively. The +dP/dt and -dP/dt, infarct size and interstitial collagen content were also improved in the NF/MNC-1D and -4D groups but not in the -7D group. Mechanistically, MNC quality and the states of systemic inflammation and damaged heart tissue influence the therapeutic efficiency of NF/MNC therapy, as revealed by another independent study using 16 pigs.</p><p>Conclusions</p><p>Injection of NF/MNC at 1 or 4 days, but not at 7 days post-MI, improves cardiac performance and prevents ventricular remodeling, confirming the importance of early intervention when using this therapy for acute MI.</p></div