Fig 1: Upregulated expression of NSE was positively correlated with distant metastasis of SCLC patients. (A) The relationship between serum NSE concentration and the prognosis of SCLC patient was evaluated by univariate and multivariate COX regression analysis. The correlations between NSE serum concentration and TNM stage (B), T stage (C), N stage (D), and M stage (E) were evaluated. (F) The correlations between NSE serum concentration and different sites of metastasis. (G) The expression difference of NSE in SCLC with or without distant metastasis was detected by immunohistochemistry (Left scale bars: 50 um, Right scale bars: 25 um). Data is represented as mean ± SD. The significance level is represented by ns, p < 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Fig 2: NSE required β-catenin to promote migration, invasion, and EMT of SCLC cells. (A-D) Western blotting and qRT-PCR assay for the expression of β-catenin, slug, E-cadherin, and N-cadherin in NSE-overexpressing H446 cells in the presence of shβ-catenin (A, B) and in NSE-silencing H69 cells in the presence of β-catenin (C, D). (E) Wound healing assay for the migration of NSE-overexpressing H446 cells in the presence of shβ-catenin (Scale bar: 100 um). (F, G) Transwell assay for the invasion and migration of NSE-overexpressing H446 cells which were further infected with virus expressing shβ-catenin (F) and NSE-silencing H69 cells which were further infected with virus expressing β-catenin (G) (Scale bar: 50 um). Data are presented as mean values ± SD of three independent experiments. *p < 0.05 using the two-sided Student's t-test. ns: no significance.
Fig 3: NSE induced EMT in SCLC cells. (A, B) NSE overexpression promoted the EMT process of SCLC. (A) Western blot assay was performed to measure the protein expression levels of the EMT-related markers (Snail, N-cadherin and E-cadherin). (B) qRT-PCR was performed to measure the mRNA expression levels of the EMT markers. (C, D) NSE knockdown represses the EMT process of SCLC. Protein levels (C) and mRNA levels (D) of EMT-related markers were measured using western blot or qRT-PCR, respectively. These results were repeated of three independent experiments.
Fig 4: NSE interacted with β-catenin and inhibited its protein degradation. (A, B) H446 cells were transfected with Flag-β-catenin or GFP-NSE or both vectors. IP was performed with anti-Flag (A) or anti-GFP (B), followed by Western blot analysis. (C) H446-EV and H446-NSE cells were cultured in cell culture medium and treated with 20 μg/ml cycloheximide (CHX) for 0, 6, 12, 18, and 24 h, and then collected total cell for western blot analysis. Data are presented as mean values ± SD. *p < 0.05 using the two-sided Student's t-test.
Fig 5: NSE promoted EMT and tumor metastasis by activating the Wnt/β-catenin pathway in vivo. H446 cells and H69 cells were transfected with Luciferase and GFP-NSE or sh1NSE or sh2NSE or vectors and then injected through the left cardiac ventricle of anesthetized nude mice. (A, B) The bioluminescence images analysis was conducted by using an IVIS imaging system in nude mice models of NSE-overexpressing H446 cells (A) and NSE-silencing H69 cells (B). (C, D) Body weight loss was observed in nude mice models of NSE-overexpressing H446 cells (C) and NSE-silencing H69 cells (D). (E, F) Kaplan–Meier plot of survival in the experiment of NSE-overexpressing group (E) and NSE-silencing group (F) (*p < 0.05, log-rank test). (G, H) qRT-PCR assay for the expression of NSE, β-catenin and EMT markers in tumor tissues of nude mice models of NSE-overexpressing H446 cells (G) and NSE-silencing H69 cells (H). Data are presented as mean values ± SD. *p < 0.05 using the two-sided Student's t-test. ns: no significance.
Supplier Page from Abcam for Anti-NSE antibody