Expression levels of COX-2, α-SMA, Col1A1, and VEGF were determined by immunoblotting after blocking with 5% nonfat milk, with goat anti-COX-2 antibody (1:500, Cayman Chemical, Ann Arbor, Michigan), a monoclonal antibody against α-SMA (Clone 1A4, 1:1000), a rabbit polyclonal antibody against Col1A1 (ORIGENE, Rockville, MD), or an anti-VEGF polyclonal antibody (1:2000, Millipore Temecula, CA), and visualized with HRP (horseradish peroxidase)-conjugated secondary antibodies using the SuperSignal West Pico Chemiluminescent substrate kit (Thermo Scientific, Rockford, IL). Monoclonal anti-GAPDH antibody (1:50,000 dilution, Sigma-Aldrich) was used as loading control.
Total RNA was isolated from cells and tumor samples using the QIAshredder and RNeasy Mini kit (Qiagen, Valencia, CA). cDNA was prepared using the iScript cDNA synthesis kit (Bio-Rad). cDNA samples were diluted 1:10 and real-time PCR was performed using IQ SYBR Green supermix and gene specific primers in the iCycler real-time PCR detection system (Quanta Bioscience, Gaithersburg, MD). All primers were designed using Beacon designer software 7.8 (PREMIER Biosoft, Palo Alto, CA). The expression of target RNA relative to the housekeeping gene HPRT1 was calculated based on the threshold cycle (Ct) as R = 2-Δ(ΔCt), where ΔCt= Ct of target - Ct of HPRT1.
Total RNA was isolated from cells and tumor samples using the QIAshredder and RNeasy Mini kit (Qiagen, Valencia, CA). cDNA was prepared using the iScript cDNA synthesis kit (Bio-Rad). cDNA samples were diluted 1:10 and real-time PCR was performed using IQ SYBR Green supermix and gene specific primers in the iCycler real-time PCR detection system (Quanta Bioscience, Gaithersburg, MD). All primers were designed using Beacon designer software 7.8 (PREMIER Biosoft, Palo Alto, CA). The expression of target RNA relative to the housekeeping gene HPRT1 was calculated based on the threshold cycle (Ct) as R = 2-Δ(ΔCt), where ΔCt= Ct of target - Ct of HPRT1.