KLF4 Time Course

A stably transfected inducible system for KLF4 was established in RKO cells as previously described (Chen et al., 2001; Chen et al., 2003). This cell line, called EcR-RKO/KLF4, contains stably transfected receptors for the insect hormone, ecdysone (EcR), and a full-length mouse KLF4 cDNA driven by a promoter that responds to ecdysone and its analogue, ponasterone A. Cells were treated with 5 µM ponasterone A (PA), for various time durations. To control for the experiment, the vehicle, ethanol, was added for the same periods of time. Keywords: Time course RNA was extracted from EcR-RKO/KLF4 cells treated with ponasterone A or vehicle control for 0, 1, 2, 4, 6, 8, 12, and 24 hr, using Trizol (Invitrogen). RNA from each time point labeled with biotin using the Enzo BioArrayTM HighYieldTM RNA Transcript Labeling kit (T7), and hybridized to Affymetrix commercial HU133A chips. Hybridization was done using the GeneChip® Fluidics Station according the Affymetrix single stain protocol. The data were scanned and analyzed by an Affymetrix GeneChip® Fluidics Station 400 scanner and Affymetrix Microarray Suite 2.