Figure 1

Characterization and differential gene expression analysis of Rasless MEFs. (A) Ras protein expression levels in MEFs treated with 4-hydroxy-tamoxifen (4OHT). Western immunoblots showing Ras protein levels after treatment with 4-hydroxy-tamoxifen (4OHT). Pan-Ras immunoblots showing the decrease in K-Ras protein expression after 4OHT treatment for 6 or 12 days of two different cell lines, DU315-6 and DU244-1. As control, 4OHT had no effect on K-Ras protein level in a representative, constitutive double knockout (H-Ras^-/-;N-Ras^-/-) MEF cell line A624-8 [21]. (B) Differential gene expression in Rasless MEFs as determined by microarray hybridizations. The Statistical Analysis of Microarray (SAM) algorithm [109] was used to identify differentially expressed probesets by comparing the microarray-generated transcriptional profiles of K-Ras^lox cell lines treated with 4OHT for 6 days (6-day 4OHT, upper left panel) or for 12 days (12-day 4OHT Rasless, upper right panel) with those of control, untreated K-Ras^lox MEFs. Only 1 differentially expressed probeset could be identified in 6-day 4OHT samples using a lax False Discovery Rate (FDR) value of 0.089 (upper left panel). In contrast, the SAM plot for the 12-day 4OHT Rasless samples allowed the identification of 3091 differentially expressed probesets using a highly stringent FDR value of 0.01 (upper right panel). Comparison of the transcriptional profile of control K-Ras^lox MEFs with those of the BRAF-rescued (lower left panel) or the MEK1-rescued (lower right panel) cells using the same FDR value of 0.01 showed that the expression of transfected BRAF or MEK1 resulted in reversal of the majority of transcriptional alterations observed in Rasless MEFs generated after 12-day 4OHT treatment since only 13 or 399 differentially expressed probesets could be identified respectively.