Baseline characteristics of primary non-muscle invasive bladder cancer patients.

<p>Baseline characteristics of primary non-muscle invasive bladder cancer patients.</p

Time to progression in non-muscle invasive bladder cancer according to <i>C16orf74</i> mRNA expression levels.

<p>Time to progression in non-muscle invasive bladder cancer according to <i>C16orf74</i> mRNA expression levels.</p

Multivariate Cox regression analysis for prediction of progression in NMIBC and in NMIBC with intravesical therapy.

<p>Tx.: Therapy, Pt.: patients, HR: hazards ratio, CI: confidence interval.</p

Anomalous Wavelength Scaling of Tightly Coupled Terahertz Metasurfaces

We theoretically and experimentally demonstrate the drastic changes in the wavelength scaling of tightly coupled metasurfaces caused by deep subwavelength variations in the distance between the unit resonators but no change in the length scale of the units themselves. This coupling-dependent wavelength scaling is elucidated by our model metasurfaces of ring resonators arranged with deep subwavelength lattice spacing <i>g</i>, and we show that narrower <i>g</i> results in rapider changes in wavelength scaling. Also, by using terahertz time-domain spectroscopy, we experimentally observed a significant shift of the spectral response arising from very small variations in lattice spacing, confirming our theoretical predictions

p21WAF1 Is Required for Interleukin-16-Induced Migration and Invasion of Vascular Smooth Muscle Cells via the p38MAPK/Sp-1/MMP-9 Pathway

<div><p>Interleukin-16 (IL-16) is a lymphocyte chemoattractant factor well known for its role in immune responses, but its role in vascular disease is unknown. Here, we explored the novel physiological function of IL-16 in vascular smooth muscle cells (VSMCs). The expression of IL-16 and its receptor CD4 was observed in VSMCs. Treatment with IL-16 enhanced the migration and invasion by VSMCs without altering the proliferative potential. IL-16 induced MMP-9 expression via the binding activity of transcription factors NF-κB, AP-1, and Sp-1 motifs in VSMCs. Among the relevant signaling pathways examined, only p38MAPK phosphorylation was significantly stimulated in IL-16-treated VSMCs. Treatment with p38MAPK inhibitor SB203580 prevented the IL-16-induced migration and invasion of VSMCs. SB203580 treatment inhibited the MMP-9 expression and activation of Sp-1 binding in IL-16-treated VSMCs, and siRNA knockdown of CD4 expression blocked the induction of migration, invasion, p38MAPK phosphorylation, MMP-9 expression, and Sp-1 binding activation stimulated by IL-16. The IL-16 induced cell-cycle-inhibitor p21WAF1 expression in VSMCs, but had no effect on the expression levels of other cell-cycle negative regulators. Finally, blockage of p21WAF1 function with specific siRNA abolished the IL-16-induced elevation of migration, invasion, p38MAPK phosphorylation, MMP-9 expression, and Sp-1 binding activation in VSMCs. Taken together, p21WAF1 was required for the induction of p38MAPK-mediated MMP-9 expression via activation of the Sp-1 binding motif, which led to migration and invasion of VSMCs interacting with IL-16/CD4. These results could provide that IL-16 is a new target in the treatment of vascular diseases such as atherosclerosis and re-stenosis.</p></div

Involvement of p21WAF1 in the induction of migration, invasion, p38MAPK phosphorylation, MMP-9 expression, and Sp-1 binding activity in IL-16-stimulated VSMCs.

<p>VSMCs were transfected with either si-p21-1 or scramble siRNA, and stimulated with IL-16 (50 ng/ml) for 24 h. (A, B) Indicated cells were examined via wound-healing migration and invasion assay. Scale bars represent 400 μm (wound-healing) and 100 μm (invasion). *P < 0.01 compared with IL-16 treatment. (C) Expression level of MMP-9 was determined by zymography and immunoblot analysis using culture medium and cell lysates. For the phosphorylation level of p38MAPK, indicated transfected cells were cultured with IL-16 for 10 min. Immunoblot was performed with antibody specific for p38MAPK using cell lysates. *P < 0.01 compared with IL-16 treatment. (D) Transfected cells were incubated with IL-16 (50 ng/ml) for 24 h. Nuclear extracts were acquired from indicated cells, then subjected to EMSA for an evaluation of the binding activity of the Sp-1 motif using radiolabeled oligonucleotide probes. One picomole of unlabeled Sp-1 was loaded as a negative control (competitor). *P < 0.01 compared with IL-16 treatment. (E) Effect of knockdown of p21WAF1 gene in VSMCs. VSMCs were transfected with either si-p21-1 or scramble siRNA. The efficacy of silencing the p21WAF1 gene was assessed by immunoblot. GAPDH was employed as a loading control. Results are reported as the means ± SE from three triplicate experiments. *P < 0.01 compared with control.</p

IL-16 induced expression of p21WAF1 in VSMCs. Quiescent VSMCs were incubated with indicated concentrations of IL-16 for 24 h.

<p>(A) Indicated cells were then subjected to flow cytometric analysis for the determination of cell-cycle distribution in the presence of IL-16. (B) The percentage of the cell population in each phase is shown in the table. (C) Cell lysates were obtained from the indicated cells, and then were subjected to immunblot using specific antibodies against p21WAF1, p27KIP1, and p53. The GAPDH was loaded as an internal control. Results are reported as the means ± SE from three triplicate experiments. *P < 0.01 compared with control.</p

Effect of si-CD4-1 on IL-16-induced promotion of migration, invasion, p38MAPK phosphorylation, MMP-9 expression, and Sp-1 binding activity in VSMCs.

<p>Cells were transfected with either si-CD4-1 or scramble siRNA, then followed by stimulation of IL-16 for 24 h. (A, B) Indicated cells were subjected to wound-healing assay and invasion assay. Scale bars represent 400 μm (wound-healing) and 100 μm (invasion). *P < 0.01 compared with IL-16 treatment. (C) Zymography and immunoblot was used to determine the MMP-9 expression using cell supernatants and cell lysates. For the phsosphorylation of p38MAPK, transfected cells were incubated for 10 min, and then the cell lysates were analyzed via immunoblot. *P < 0.01 compared with IL-16 treatment. (D) After transfection of either si-CD4-1 or scramble siRNA, indicated cells were cultured with IL-16 (50 ng/ml) for 24 h. Binding activity of the Sp-1 motif was examined by EMSA from nuclear extracts using radiolabeled oligonucleotide probes. Unlabeled Sp-1 (1 picomole) was used as a competitor control. *P < 0.01 compared with IL-16 treatment. (E) The efficacy of silencing gene of CD4 in VSMCs. Cells were transfected with either si-CD4-1 or scramble siRNA. The protein level of CD4 was evaluated by immunoblot. GADPH was used as an internal control. Results are reported as the means ± SE from three triplicate experiments. *P < 0.01 compared with control.</p

Expression of IL-16 and its receptor CD4 in VSMCs.

<p>(A, B) IL-16 and CD4 are expressed in VSMCs. Cells were cultured with DMEM medium containing 10% FBS for 24 h. Then, protein levels of IL-16 and CD4 were examined by immunoblot analysis. Lysates from rat brain tissue and rat spleen used as positive control. GAPDH expression served as the loading control. (C) Confocal staining of IL-16 in VSMCs. Cells were stained with antibody against QD565-conjugated IL-16 (red). Both DAPI and tubulin-Alexa488 were used to counterstain the nuclei (blue) and cytoplasm (green). Scale bars represent 25 μm. (D) IL-16 did not affect the proliferation of VSMCs. Subconfluent cells were cultured with serum-free medium for 24 h, prior to stimulation with IL-16 for indicated concentrations. The level of thymidine uptake was evaluated using a liquid scintillation counter. Results are represented as the means ± SE from three triplicate experiments. *P < 0.01 compared with control.</p

IL-16 induced the phosphorylation of p38MAPK in VSMCs.

<p>(A) Quiescent VSMCs were incubated with IL-16 (50/ng/ml) for the indicated time periods. Cell lysates were assessed by immunoblot analysis using specific antibodies against phopho-ERK1/2, ERK1/2, phospho-p38, p38, phospho-JNK, and JNK. Results are reported as the means ± SE from three triplicate experiments. *P < 0.01 compared with control. (B) Quiescent VSMCs were cultured with IL-16 for 10 min at the indicated concentrations. Cell lysates were subjected to immunoblot analysis for the phosphorylation of p38MAPK. *P < 0.01 compared with control (C) Quiescent VSMCs were pre-incubated with SB203580 (10 μM) for 40 min prior to IL-16 treatment (50 ng/ml) for 10 min. The phosphorylation levels of p38MAPK were evaluated by immunoblot. *P < 0.01 compared with IL-16 treatment.</p