Cells with annexin V (+) and PI (−) were deemed GDC-0068 cell line early apoptotic cells. Cells with both annexin V (+) and PI (+) were deemed late apoptotic cells. TUNEL assay To identify apoptosis in the transfected cells, we utilized the dead-end colorimetric TUNEL system kit (Promega, Madison, USA) to measure DNA fragmentation and caspase-3 activation in the GKN1 transfected cells, according to the manufacturer’s instructions. Briefly, cells were fixed in 4% paraformaldehyde solution for 25 min at room temperature, rinsed in PBS, and permeabilized by incubating the slides in 0.2%
Triton X-100 solution. Cells were then incubated with a terminal deoxynucleotidyl transferase (TdT) reaction mixture containing biotinylated nucleotides and TdT at 37°C for 60 min, and rinsed with 1 × SSC (sodium chloride-sodium citrate buffer) and PBS. Next, streptavidin HRP was added to the cells, and the cell
slides were stained with 3,3′-diaminobenzidine color solution. Finally, cells were examined under a light microscope and the number of positive cells was counted and summarized from a total of 10 high power fields. CP673451 in vivo Cell cycle analysis To analyze cell cycle distribution, transfected cells were grown and treated with 25 M olomoucine (Santa Cruz Biotechnologies, Santa Cruz, USA) for 1 h, and then incubated with regular culture medium for an additional 1 h . Cells were then collected and subjected to cell cycle analysis by flow cytometry as described in the previous section. Captisol concentration Sensitivity to 5-FU treatment To detect the role of GKN1 in mediating sensitivity of gastric cancer cells to 5-FU Amisulpride treatment, we grew and treated GKN1 transfected tumor cells with 5-FU (Sigma) or DMSO for 24 h and 48 h. Concentrations of
5-FU ranged from 0.25 to 1.0 mmol/L. The apoptosis rate from these cells was detected by flow cytometry as previously described. cDNA microarray analysis To perform cDNA microarray analysis, total cellular RNA from GKN1-transfected and vector-control tumor cells were isolated with the Trizol® Reagent (Invitrogen). RNA was then reversely transcribed into cDNA using the TrueLabeling-AMP Linear RNA amplification kit (Superarray, Frederick, MD, USA), and then converted into biotin-labeled cRNA using biotin-16-UTP and an in vitro transcription kit (Roche, Basel, Switzerland). The newly synthesized cRNA probes were then purified with the ArrayGrade cRNA cleanup kit (Superarray) and then added to the pretreated Oligo GEArrays Human Apoptosis Microarray (OHS-012 from Superarray) that contains 112 apoptosis-related genes. The microarray was then hybridized overnight at 42oC. The next day, the hybridized arrays were washed and detected by chemiluminescence according to the manufacturer’s instructions (Pierce). The data were analyzed using GEArray Expression Analysis software (Superarray). If spot intensity increased by more than two fold, this gene was deemed upregulated.