ith pri mary followed by horseradish pero idase conjugated secondary antibodies. The primary antibodies used in this study are listed in selleck chem Additional file 1 Table S2. The membranes were developed using an ECL developing so lution followed by autoradiography. All the e periments were performed at least three times inde pendently and that typical results were shown. In each sample, the protein e pression shown in each band was quantified after normalization to the GAPDH e pression level. The error bars shown in the relevant figures indi cated the standard deviation of the quantification results in all e periments. Luciferase reporter assay for the NME4 3 UTR The pMIR REPORT firefly luciferase vector plasmid was used. The 3 UTR region of wide type NME4 was amplified by PCR and cloned downstream of the luciferase vector.

A mutant sequence was also cloned as a validation plasmid. pMIR, p UTR WT, and p UTR mut was co transfected with the miR 196 specific antagomirs or overe pression plasmids into OECM1 or SAS cells. The pRL SV vector containing Renilla luciferase was also transfected for each condition as a reference control. Firefly and Renilla luciferase activities were measured using the Dual Luciferase Reporter Assay Sys tem according to the manufacturers instruc tions. All the e periments were performed triplicate for at least three times, and the similar results were ob tained. The error bars shown in the relevant figures indi cated the standard deviation of a triplicate e periment. Immunofluorescence staining and confocal microscopy Immunofluorescence staining and confocal microscopy were performed as previously described.

Briefly, cells were seeded onto coverslips coated with poly L lysine and incubated overnight at 37 C. After washing, the cells were fi ed with formaldehyde, permeabilized with a permeation buffer, and blocked with 1% FBS. After overnight incubation with primary antibodies, the coverslips were incubated with fluorescence conjugated secondary antibodies. The coverslips were then mounted with mounting medium containing DAPI dye, and the fluores cence was visualized using a confocal laser microscope. Patients and clinical association study and statistical analysis This study was approved by the Institutional Review Broad of the Human Investigation Committee in Chang Gung Memorial Hospital.

Written informed consent was obtained from all patients participating in this study. Cilengitide Fifty four patients who visited Chang Gung Memorial selleck chem inhibitor Hospital were recruited for this study. The characteristics of these patients are summarized in Table 1. This study consisted of 5 females and 49 males. The mean age of the patients was 54. 6 years old, with a median age of 53. 0 years. A total of 25 patients consumed alcohol, 30 patients smoked cigarettes, and 37 chewed betel quid. Cancer lesions were in oral tongue, buccal mucosa, other oral cavity sites, or soft plate. The surgically dissected cancer tissues and small pieces of adjacent normal counterpart were