ABSTRACT: Confluent human umbilical vein endothelial cells (HUVECs) were exposed to Insulin (100 micM) for 2 hours. Ribosomal profiling via gradient centrifugation and fractionation was used to separate monosome, or under-translated, and polysome, or actively-translated, mRNA species that were then used to probe cDNA arrays, a process known as Translation State Array Analysis (TSAA). Four samples were obtained from these experiments: control monosome, control polysome, Insulin monosome and Insulin polysome. using the normalized signal intensities from the GeneFilters, we calculated a translation index, or measure of movement of an mRNA molecule from the monosome to the polysome fraction upon stimulation. This calculation was made as follows: (Insulin polysome/Insulin monosome)/(control polysome/control monosome). Translational indices greater than 2.5 (upregulated) or lower than 0.4 (downregulated) were chosen for further study. HUVECs were stimulated with Insulin (100 micM) for 2 hours. Cycloheximide (CHX) was added to a final concentration of 100 ng/mL for 5 minutes and cells were then rinsed with cold HBSS containing CHX (100 ng/mL) and scraped from the dish and pelleted by centrifugation (2000 x g, 5 minutes). The supernatant was removed and cells were carefully resuspended in 375 ïL Low Salt Buffer (LSB; 20 mM Tris, 10 mM NaCl, 3 mM MgCl2, pH 7.4) + RNasin (40 U/mL) + DTT (10 nM) for 3 minutes on ice. 125 ïL LSB Lysis buffer (LSB containing 200 mM sucrose and 1.2% Triton-X100) was then added and cells disrupted by pipetting. The mixture was then transferred to 1.7 mL microfuge tubes and centrifuged (20,000 x g, 1 minute, 4ï° C) to remove nuclei and cellular debris. The supernatant was transferred to a new tube containing 50 ïL LSB + RNasin (40 U/mL) + DTT (10 nM) and 15 ïL 5M NaCl. This mixture was then layered onto prepoured gradients (15-50% sucrose in LSB). Gradients were spun at 43,700 rpm for 90 minutes (4ï° C) and then run on a density gradient flow cell fractionator (Isco) to obtain ribosomal profiles. Ribosomal fractions corresponding to monosomes (M) and polysomes (P) (Figure 1) were collected into Trizol LS (Invitrogen) and total RNA isolated according to the directions of the manufacturer. Total RNA was reverse transcribed using oligo dT primers in the presence of 33P dCTP to generate labeled cDNA probes from the M and P associated mRNA fractions of control and stimulated cells and these probes used to detect specific array elements on four identical Research Genetics (ResGen) Named Human Gene cDNA GeneFilters (GF 211, Invitrogen). These filters were washed using high stringency conditions and exposed to phosphorimaging screens for analysis using Pathways 3, proprietary software included with the ResGen filters. Hybridization signals were normalized to the average intensity of the hybridization signals from the particular array filter to correct for differing global hybridization efficiencies between filters. To determine if a particular hybridization signal was significantly above background levels, an initial screen of the array elements was conducted by dividing the hybridization signal of an individual cDNA spot by the background signal on particular filter. Background signal levels were determined using the Pathways 3 analysis software that measures the signal intensity of the area between cDNA spots and averages that signal over the area of the entire array. Array elements were considered to be distinguishable from background if the hybridization signal/background ratio was greater than 1.1. Signal intensities from each filter were normalized by dividing the raw signal intensity of the cDNA spot by the average signal intensity for the entire filter. The translation state (TS) of each qualifying array element was determined by dividing the normalized signal intensity of the element in the polysome fraction by the normalized signal intensity of the element in the monosome fraction: TS=P/M. The Translation Index (TLI), a measure of the change of translation states of individual genes, was determined by calculating the ratio of the treated cell TS to the control cell TS; TLI = TSe/TSc where e represents experimental or treated conditions and c represents controls. In this scenario, a gene that is not regulated by translation would have a TLI value of 1, actively translated genes would have a TLI value greater than 1, whereas a poorly translated or under-translated gene would have a TLI value less than 1. Arbitrary TLI values of 2.5 and 0.4 were used to categorize individual array elements as being translationally regulated.
