Cell growth curve Exponentially growing normal and transformed IEC-6 cells were
cultivated in 96-well plate, with 1 × 104 cells in each well. Twelve hours later,3H-TdR 7.4 × 104Bq/ml was added into the culture media, and the plate was returned to the incubator for further cultivation. Cells were washed with cold PBS after discarding the #CB-839 molecular weight randurls[1|1|,|CHEM1|]# culture media at indicated time. Excess3H-TdR was removed by washing with 3 ml PBS. The cells were resuspended in 10% trichloroacetic acid (TCA) with vigorous vortexing. The cellular lysates were vacuum-filtered and then washed with cold 5% TCA. Incorporated3H-TdR was measured in a liquid scintillation counter (Beckman LS5000TA, Fullerton, California, USA). The procedures were performed 3
selleck chemicals llc times in duplicate 24-well culture dishes. Values are expressed as mean ± SEM. Gene expression studies using Rat Oligo GEArray A rat Oligo GEArray microarray (Exiqon, Denmark) was employed to detect altered gene expression associated with cell transformation. RNA preparation: Total RNA was isolated from the cells of each group using TriPure reagent kit according to the manufacturer’s protocol (Roche Diagnostics Co.). The integrity of RNA sample was assessed by viewing the ethidium bromide-stained 28 S and 18 S ribosomal RNA bands, and the purity of RNA sample was verified by the absorption ratio OD260 nm/OD280 nm. Equal amounts of RNA isolated from normal and transformed IEC-6 cells were pooled for Erastin in vitro the following microarray detections. 3 μg total RNA was reverse transcribed into Biotin-16-dUTP-labeled cDNA probes with the TrueLabeling-AMP method according to the manufacturer’s instructions. The microarray membranes were pre-hybridized at 60°C for at least 2 h. Hybridization of the Biotin-labeled cDNA probes to the membranes was carried out at 60°C overnight with slow agitation in a hybridization oven. The hybridized membranes were washed in saline sodium citrate buffer. Then membranes were incubated with alkaline phosphatase-conjugated streptavidin,
washed and incubated with the chemiluminescent substrate CDP-Star. Images of the membranes were acquired using the Chemidoc XRS system (Biorad Laboratories) and analyzed. The relative expression level of each gene was determined by comparing the signal intensity of each gene in the array after correction for background and normalization. microRNA chips miRCURY LNA™ microRNA chips (Exiqon, Vedbaek, Denmark) were employed to detect altered miRNA expression associated with cell transformation. The chips (version 9.2) contained totally 2056 probes, including human, mouse and rat miRNA genes, in triplicate. Total RNA (2–4 μg) was 3′-end-labeled using T4 RNA ligase and a Cy3-labeled RNA linker by the following procedure: RNA in 2.0 μL of water was combined with 1.0 μL of CIP buffer and CIP (Cat#208021, Exiqon). The mixture was incubated for 30 min at 37°C, and was terminated by incubation for 3 min at 80°C. Then 3.