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A total of 20 mg protein lysates were separated by SDS-PAGE and then the target proteins were detected by Western blot analysis with the following antibodies: rabbit anti-human Wnt2 monoclonal antibody (Abcam), rabbit anti-human ��-catenin monoclonal antibody (Cell signaling Technology) and mouse anti-human ��-actin monoclonal antibody (Abmart). ELISA A human Wnt2 ELISA kit (CUSABIO) was used to detect Wnt2 concentration in the serum samples or the supernatants of cell cultures. Colony formation assay and 5-ethynyl-2��-deoxyuridine (EdU) incorporation assay Colony formation assay was carried out as described previously [16]. In brief, six-hole plate was seeded at a density of 800 cells per hole. Plates were maintained at 37��C in humidified incubator and culture medium was replaced every 3 days. After 2 weeks, Midostaurin cells were stained with 0.5% Crystal Violet and the average colony number of five random fields (4 �� in magnification) was counted under microscope. A EDU incorporation kit (Ribobio) was used to measure the DNA synthesis rate as described [17]. All experiments were conducted in triplicates. Representative views were photographed. In vivo assay H1299 cells transfected with Wnt2 expression vector or empty vector were suspended in phosphate buffer solution. Then the cell PRDX5 suspension were injected subcutaneously into the inguinal region of 6 pairs of 4-week-old male Balb/c nude mice (Vital River Laboratory Animal Technology Co. Ltd, China). Tumor diameter was measured every week until the mice were anesthetized and sacrificed at the end of week 6. Tumor volume was determined by the longest and shortest diameter of the tumor and calculated as follows: volume = (shortest diameter)2 �� (longest diameter) �� 0.5 [18]. The animal handling and all experimental procedures were approved by the Animal Ethics Committee of TJMUCH. Statistical analysis Statistical analyses were carried out by the IBM SPSS Statistics Program. Each experiment was done in triplicate and values are presented as mean �� SD. A Student t test or ANOVA for unpaired data was used to compare mean values. Kaplan-Meier curves were calculated for relevant variables and Wnt2 expression; The log-rank test was used find more to analyze the differences in survival times among patient subgroups. The risk factors associated with the prognosis of these patients were evaluated by Cox��s proportional hazard regression model. The Forward: LR procedure was used for the univariate analysis. All probability values had a statistical power level of 90% and a 2-sided level of 5%. Serum Wnt2 levels before and after surgery were analyzed by the paired t test. Receiver operating characteristic (ROC) curve analysis was performed in MedCalc Program to compare Wnt2 and CEA for their role in predicting NSCLC. P