Template and 1.0 l each of the forward and PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28192408 reverse primers (10 M) (see Additional file 1). The PCR was done under the conditions at 94 for 30 seconds, then 40 cycles of amplification at 94 for 30 seconds, 60 for 30 seconds. Each gene was normalized to the internal -actin levels. Each sample was run in triplicate to ensure quantitative accuracy, and the threshold cycle numbers (Ct) were averaged. The results were reported as tumor tissues: normal tissues (T:N) ratios and calculated using the 2-Ct method [14].Western blotImmunofluorescence analysis was done on the cultured cells. Briefly, the cells grown on coverslips were fixed in -20 acetone for 10 minutes and then incubated with the rabbit anti-ERGIC3 polyclonal antibody (Abcam), and anti-MUC1 mouse monoclonal antibody (mAb) A76A/C7 (Glycotope, Berlin, Germany), anti-ST mAb [15], anti-calreticulin mAb (Abcam), and anti-58K Golgi protein mAb (Abcam) at 4 overnight. Following the washes, the cells were incubated with FITC-coupled anti-rabbit antibody (BD sciences, Franklin Lakes, NJ, USA) or with Cy3coupled anti-mouse antibody (Millipore, Billerica, MA, USA) for 1 hour. Subsequently, the nuclei were finally counterstained with diamidinophenylindole. The slides were analyzed under confocal laser scanning microscopy.Construction of the expression vector and the cell transfectionTotal protein was extracted from cultured cells and tissues, separated by electrophoresis, and then transferred into PVDF membrane according to the routine protocol. The blotted membrane was incubated with the rabbitTo generate the plasmid expressing ERGIC3, we cloned the open reading frame of ERGIC3 and used the following specific primers (upper case, restriction enzyme sequences): forward, 5′-GGTGGTGAATTCatgaggcgctg gggaagct-3′; reverse,5′-GGTGGTGGATCCaccgaggagggt gactacgttgtctt-3′. After running PCR, the product was cut by EcoR I and BamH I. The purified DNA was ligated into the pLXSN expression vector (Clontech Corp.). The empty vector served as a control. The vectors were transfected into BEAS-2B cell by PX-478 solubility lipofectamine LTX and PLUS (Invitrogen Corp.) according toWu et al. BMC Cancer 2013, 13:44 http://www.biomedcentral.com/1471-2407/13/Page 4 ofthe instructions. The over-expression of ERGIC3 was validated by q-RT-PCR and western blot.Gene silencing by RNA interferenceusing IBM SPSS 19 (SPSS inc., Chicago, Illinois). Differences were considered significant if P < 0.05.ResultsGeneration of SSH cDNA librariesFor gene silencing of ERGIC3, pGPU6/GFP/NeoERGIC3-shRNA and its control vector, pGPU6/GFP/ Neo-NC-shRNA, were used (Jima Corp., Shanghai, China). Briefly, the oligonucleotide GGAGGACTATC CAGGCATTGT was designed to interfere specifically with ERGIC3 gene expression. As a negative control, we used the oligonucleotide GTTCTCCGAACGTGT CACGT, which has no significant match in a BLASTn search (human NCBI nr database). The vectors were transfected into GLC-82 cells by lipofectamine LTX and PLUS. The gene silencing of ERGIC3 was validated by q-RT-PCR and western blot.Cell proliferation assayCell proliferation analysis was based on the capacity of mitochondrial enzymes to transform MTT to MTT formazan. More succinctly, after being transfected for 48 hours, GLC-82 and BEAS-2B were collected and transferred into 96-well plates (4*103 cells/well), then all cells were treated with MTT (5 mg/ml) every 24 hours. 10 l of MTT was added to each well and cells were incubated at 37 for 2 hours. Then, the culture medium with.