Antibodies and Reagents
The polyclonal antibody directed against FAP-1 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and the anti-mouse Actin antibody was purchased from Sigma (St Louis, MO). Temozolomide was purchased from Roche. FAP-1 siRNA and control siRNA were purchased from Santa Cruz Biotechnology.
Cells and Cell Culture
U87MG and LN18 cells purchased from ATCC were maintained in Dulbecco’s Modified Eagle’s Medium containing 10% fetal bovine serum (FBS) (DKSH, Hallam, Victoria, Australia), 2mM glutamine, 100U/ml penicillin and 100 µg/ml streptomycin (Invitrogen, Frederick, MD), and incubated in a humidified atmosphere containing 10% CO2 at 37°C.
SiRNA Knockdown
To assess the effects of FAP-1 knockdown, FAP-1 siRNA or control siRNA was transiently transfected into cells using metafectene™, and then the cells were seeded into appropriate tissue culture plates.
Cell Viability Assays. Cells were plated in 96-well plates and allowed to adhere overnight. Triplicate wells were treated with varying concentrations of temozolomide with or without radiation for 3-7 days. Cells were then lysed and cell viability was determined using a commercially available Cell Titer-Glo kit (Promega) following the manufacturer’s instructions. Samples were read on a bioluminometer.
Western Blot Analysis
Cells were lysed with lysis buffer (containing 50mM Tris (pH 7.4), 150mM NaCl, 1% Triton-X-100, 50mM NaF, 2mM MgCl2, 1mM Na3VO4 and protease inhibitor cocktail (Roche)) and clarified by centrifugation (13,000g for 15 min at 4°C). Proteins were then separated by SDS-PAGE (Invitrogen), blotted onto nitrocellulose and probed with the indicated primary antibodies. The signal was visualized using the ECL chemiluminescence detection kit (GE Healthcare, Rydelmere, N.S.W., and Australia) following incubation with appropriate secondary antibodies.
Migration Assays
Migration experiments with FAP-1 siRNA knockdown were carried out using xCELLigence RTCA DP instrument (Roche Diagnostics GmbH, Germany) as per the manufacturer’s instructions. Briefly, cells were transfected with FAP-1 and control siRNA and allowed to adhere onto tissue culture plates for 2 days before they were classified and seeded into CIM16 plates for the migration assay. Migration was analyzed after 2-3 days of incubation in CIM16 plates.
Data mining using Oncomine. Gene expression of 16 phosphatases was analyzed using Oncomine 4.4.4.3 (www.oncomine.org, Compendia BioscienceTM, Ann Arbor, MI, USA, part of Life Technologies), an online tool that acquires 715 mRNA and copy number expression datasets from 86,733 cancer and normal tissue samples. These datasets are compiled from publically available cancer microarray data, which are processed according to the same criteria before being made available. We used the Oncomine compendium to study the expression profiles of 16 different phosphatases in brain cancer tissue versus their normal tissue counterparts by subjecting each dataset to threshold criteria for analysis. A p-value<0.05 and an mRNA expression fold change >1.4 were taken as the initial threshold criteria in this study. The fold change is classified as the difference between mRNA expression levels of genes, especially those of interest, in the cancer tissue and in the normal tissue. Based on the threshold criteria, Oncomine will then assign a gene rank percentile for each gene studied in a dataset. This figure is the percentage rank of your gene of interest calculated by comparing its p-value with the p-values of all the other genes within the same dataset. The gene expression data generated through Oncomine were log transformed and standard deviation was normalized to each array studied.
Statistical Analysis
Graph and statistical analysis was performed using Graph Pad Prism 6 (Graph Pad, San Diego, CA) or EXCEL. Statistical significance was determined by using ANOVA or Students’ T-tests.