br Construction of stable cell lines br AGS and
2.3. Construction of stable cell lines
AGS and SGC7901 cell lines stably expressing ENO1-specific short hairpin RNA (shRNA) and plasmid encoding human ENO1 were gen-erated using a lentivirus shRNA technique (GenePharma, Shanghai, China), as described in our previous study (Yao et al., 2016). Empty lentivirus vector infected stable cell lines were also produced by the above protocols. The human ENO1 shRNA target sequences are 5′-GGA GAA AUA UGG GAA AGA UTT-3′.
2.4. Protein extraction and Western blot analysis
Briefly, cells were washed with cold PBS (Gibco) and were lysed in ice-cold RIPA lysis buﬀer (Beyotime Inc, NanTong, China) supple-mented with cocktails of protease and phosphatase inhibitors (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) according to manufac-turer's protocol. Total proteins were separated by 8% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF; Millipore, Billerica, MA) membranes, which was blocked with 5% non-fat milk with 5% skimmed milk in TBS/0.1% Tween for 1 h at room temperature. The membranes were then incubated with the indicated primary Fluxametamide at 4 °C with gentle shaking overnight, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies diluted using PBS (dilution, 1:5000; OriGene Technologies, Inc., Rockville, MD, USA) for 1 h at room temperature. Then the proteins were visualized using chemiluminescence kit (EMD Millipore, Billerica, MA, USA) and signals were quantified by ImageJ software (version 1.46; National Institutes of Health, Bethesda, MD, USA). Detailed protocol was European Journal of Pharmacology 845 (2019) 8–15
Relationships between ENO1 expression and clinicopathological features in 76 patients with GC.
Clinic parameters Case no. ENO1 expression χ2 P value
None or low High
Depth of invasion
P-values < 0.05 were considered statistically significant.
2.5. Colony formation assay
In total, 1000 cells were placed in 6-well plates for 7–10 days, and then fixed with 4% paraformaldehyde (Beyotime Institute of Biotechnology; Haimen, China) for 10 min at room temperature and stained with 0.1% crystal violet (Beyotime Institute of Biotechnology) for 15 min at room temperature. The number of foci containing > 100 cells was determined at 40x magnification using an optical microscope (Nikon Corporation; Tokyo, Japan), and the images were captured by a digital camera (Nikon Corporation; Tokyo, Japan). Detailed protocol was described in our previous study (Sun et al., 2018a, 2018b).
2.6. Cell migration assay
GC cell migration was assessed using Transwell chambers (pore size, 8.0 µm; Corning Inc., Corning, New York, USA). Cell suspensions (200 µl containing 50,000 cells) were seeded onto the filters in 24-well chambers and 750 µl medium containing 10% FBS was placed in the lower chambers as a chemoattractant. The cells were allowed to mi-grate for 48 h at 37 °C and cells remaining on the upper surface of the membrane were then removed using a cotton swab. The filters were fixed with 4% paraformaldehyde (Beyotime Institute of Biotechnology) for 10 min at room temperature, and the cells were stained with 0.1% crystal violet solution (Beyotime Institute of Biotechnology) for 15 min at room temperature. The cells that had migrated from the upper to the
Name Company Catalog number Antibody concentration
lower side of the filter were counted in 5 randomly selected fields per sample using a light microscope (magnification, 200×; Nikon Corporation). Detailed protocol was described in our previous study (Sun et al., 2018a, 2018b).
2.7. Statistical analysis
Data are presented as mean ± standard error of the mean (S.E.M). Statistical significance was analyzed using a Student t-test (paired or unpaired, two-tailed) or one-way analysis of variance followed by the Student-Newman-Keuls test. Pearson χ2 test was used to analyze the relationships between ENO1 expression and clinicopathologic factors. A value of P < 0.05 was considered to indicate a statistically significant diﬀerence.
3.1. ENO1 expression is upregulated in human GC and is associated with lymph node metastasis and TNM stage
Previous studies (Qian et al., 2017; Qiao et al., 2018; Liu et al., 2015) have showed that ENO1 is overexpressed in GC and ectopic ENO1 expression predicted a poor clinical outcome. To further in-vestigate the clinical significance of ENO1 in gastric carcinoma devel-opment, we first detected ENO1 expression in 18 randomly selected pairs of human GC tissues and adjacent nontumorous tissues by Western blotting. Our results showed that ENO1 expression was higher in most primary GC tissues than that in the matched paraneoplastic tissues (Fig. 1A and B).