NLK c-Myb expression and clinicopathological parameters in 62 breast cancer specimens.

<p>Statistical analyses were performed by the Pearson χ2 test.</p>*<p>P<0.05 is considered significant.</p

Effect of NLK overexpression on apoptosis of MCF-7 cells.

<p>(A). MCF-7 cells were transfected with pcDNA3.1-EGFP, pcDNA3.1-EGFP-NLK or nothing (control). Western blotting was performed using anti-active-caspase-3 and anti-β-actin antibodies. The data are presented as the mean±standard error of three experiments. (B). The effect of NLK overexpression on apoptosis was assessed by Annexin V and PI staining; cells that were double stained with Annexin V and PI were considered to be apoptotic (lower right). Flow cytometry confirmed the induction of apoptosis in MCF-7 cells, for which overexpression of NLK caused an increase in apoptosis. (C). MCF-7 cells were transfected with pcDNA3.1-EGFP, pcDNA3.1-EGFP-NLK or nothing (control). Forty-eight hours after transfection, DAPI-stained nuclei were visualized by fluorescence microscopy, and images were taken. The bar chart shows the ratio of necrotic to transfected cells. The data are represented as the mean ± SEM (n = 3, *, #, P<0.01, compared with control).</p

NLK induces c-Myb degradation in MCF-7 cells.

<p>(A). MCF-7 cells were transfected with the pcDNA3.1-Myc-NLK or pcDNA3.1-Myc expression vectors, and 32 hours posttransfection, 26S proteasome inhibitor MG132 and DMSO as control were added for 16 hours. At 48 hours posttransfection, whole cell lysates were prepared, and proteins were analyzed by immunoblotting. The data were representative of three experiments. (B). pcDNA3.1-Myc-NLK or empty vector was transfected into MCF-7 cells using Lipofectamine 2000. Western blotting was performed using antibodies against c-myc, Bcl-2 and β-actin. The bar chart demonstrates the ratio of c-myc and Bcl-2 proteins to β-actin as determined by densitometry. The data are represented as the mean ± SEM (n = 3, *, #, P<0.01, compared with control).</p

Expression of NLK and c-Myb in human breast cancer.

<p>Paraffin-embedded tissue sections were stained with antibodies for NLK, c-Myb and Ki-67 and then counterstained with hematoxylin. Fig. A–C, E–G High NLK expression was observed in benign breast disease and breast carcinoma specimens (grade I), whereas c-Myb and Ki67 levels were low in the same specimens (SP×400). Fig. I-K High levels of c-Myb and Ki67 were observed in grade III tumor cells. In contrast, NLK expression was low. Fig. 1D, H, and L show negative controls for the benign breast disease and the breast carcinoma specimens. Experimental details are described in the Materials and Methods section. (M) Expression of NLK and c-Myb in eight representative paired samples of breast carcinomas and adjacent normal tissues. (N) Western blot analysis of endogenous NLK and c-Myb in a normal human breast epithelial cell line (HBL-100) and two human breast cancer cell lines (MDA-MB-231 (ER–) and MCF-7 (ER+)). β-actin was used as a loading control. The experiment was repeated at least three times. (O) Quantification indicated that MDA-MB-231 cells displayed the highest levels of NLK and the lowest levels of c-Myb among the two tumor cell lines. In contrast, the lowest NLK and highest c-Myb expression were observed in MCF-7 cells.</p

Graphic representation of the relationship between NLK and Ki-67 expression in breast carcinoma.

<p>Scatterplot of NLK versus Ki-67 levels with a regression line showing the correlation between the two levels using the Spearman’s correlation coefficient (<i>P<</i>0.05).</p

NLK plays an antiproliferative role in breast cancer cells.

<p>(A) Flow cytometry analysis of cell cycle progression in MCF-7 cells. Cells that were synchronized at G1 progressed into the cell cycle 0, 2, 6, 12, and 24 hours after serum stimulation. Finally, most of the cells entered S phase. (B) MCF-7 cells were serum starved for 48 hours (S48h); upon serum stimulation, cell lysates were prepared and analyzed by Western blotting using antibodies against NLK, c-Myb and β-actin. β-actin was used as a control for loading and protein integrity. (C) The bar graph demonstrates the ratio of NLK and c-Myb proteins to β-actin at each time point, as determined by densitometry. The data are represented as the mean ± SEM (n = 3, *, #, P<0.01, compared with control: S48h). S: serum starvation; R: serum stimulation. (D). Light microscopy showing that pcDNA3.1-EGFP and pcDNA3.1-EGFP-NLK were expressed in MCF-7 cells. Whole cell extracts were prepared 2 days after the transfection. Proteins were analyzed by immunoblotting using an anti-EGFP antibody. (E). MCF-7 cells were transfected with pcDNA3.1-EGFP-NLK or nothing (control). Western blotting was performed using anti-PCNA and anti-β-actin antibodies. The data are presented as the mean±standard error of three experiments. (F). MCF-7 cells were transfected with pcDNA3.1-EGFP, pcDNA3.1-EGFP-NLK or nothing (control). Cell growth of the transfected cells was assessed by the MTT cell viability assay. Four hours after transfection, cells were maintained in complete media, and cell growth was determined by MTT assay at each indicated time point. The data are presented as the mean±standard error of three experiments. (G). Cell cycle analysis was performed by staining NLK overexpressing MCF-7 cells with PI. MCF-7 cells were transfected with pcDNA3.1-EGFP, pcDNA3.1-EGFP-NLK, or nothing (control). Forty-eight hours after transfection, cells were trypsinized, fixed in 70% alcohol, and incubated for 30 minutes in PBS containing 10 mg/ml of RNase A at 37°C. After the incubation, cells were stained with 5 mg/ml PI.</p

Table_1_Nomogram predicting overall survival after surgical resection for retroperitoneal leiomyosarcoma patients.docx

BackgroundSurgery is the best way to cure the retroperitoneal leiomyosarcoma (RLMS), and there is currently no prediction model on RLMS after surgical resection. The objective of this study was to develop a nomogram to predict the overall survival (OS) of patients with RLMS after surgical resection.MethodsPatients who underwent surgical resection from September 2010 to December 2020 were included. The nomogram was constructed based on the COX regression model, and the discrimination was assessed using the concordance index. The predicted OS and actual OS were evaluated with the assistance of calibration plots.Results118 patients were included. The median OS for all patients was 47.8 (95% confidence interval (CI), 35.9-59.7) months. Most tumor were completely resected (n=106, 89.8%). The proportions of French National Federation of Comprehensive Cancer Centres (FNCLCC) classification were equal as grade 1, grade 2, and grade 3 (31.4%, 30.5%, and 38.1%, respectively). The tumor diameter of 73.7% (n=85) patients was greater than 5 cm, the lesions of 23.7% (n=28) were multifocal, and 55.1% (n=65) patients had more than one organ resected. The OS nomogram was constructed based on the number of resected organs, tumor diameter, FNCLCC grade, and multifocal lesions. The concordance index of the nomogram was 0.779 (95% CI, 0.659-0.898), the predicted OS and actual OS were in good fitness in calibration curves.ConclusionThe nomogram prediction model established in this study is helpful for postoperative consultation and the selection of patients for clinical trial enrollment.</p

Impoundment-induced nitrogen–phosphorus imbalance in cascade reservoirs alleviated by input of anthropogenic nutrients

<p>The ratio of nitrogen to phosphorus (N:P) is an important variable that has a close relationship with the ecological problems of nuisance algal blooms and eutrophication in aquatic environments in terms of nutrient limitation. Reservoirs generally have much higher retention efficiency for P than for N. This inherent dissimilarity in the N and P biogeochemical cycles likely results in N–P stoichiometric imbalance in downstream rivers and reservoirs, consequently causing an increase in the N:P ratio and aggravating P limitation. Here we determined the total N (TN) and total P (TP) concentrations in the cascade reservoirs of the Wujiang River and Lancangjiang River basins. The results show that TN:TP ratios in these 2 basins exhibited a common inverted V-shaped (∧) pattern downstream. We found that P is not only retained by reservoirs more efficiently than N but is also replenished at faster rates than N given anthropogenic impacts; consequently, the N–P imbalance caused by these impoundments is alleviated within a short distance downstream because of inputs of anthropogenic nutrients. Our research suggests that construction of cascade reservoirs does not necessarily lead to strict P deficiency and anomalously high N:P ratios downstream.</p