Figure 5

Differential expression of microRNAs in Rasless cells. (A) Statistical identification of differentially expressed miRNAs in Rasless MEFs. SAM contrasts [109] comparing the microarray-generated miRNA expression profiles of K-Ras^lox cell lines treated with 4OHT for 6-days (left panel) or for 12-days (right panel) with those of control, untreated K-Ras^lox MEFs. The plots identified 11 differentially expressed miRNAs after 6-days of 4OHT treatment (left panel), and 103 differentially expressed miRNAs after the 12-day treatment (right panel) using similar FDR = 0.1values. Differential expression for a given miRNA is calculated by the distance of the spot representing its expression value to the no-change diagonal. Green dots depict differentially expressed miRNAs. Black dots remaining close to the diagonal represent miRNAs without significant expression changes relative to the control samples. (B) Hierarchical clustering of differentially expressed microRNAs of Rasless MEFs. Heatmap generated by cluster analysis of the absolute expression values of the group of 103 differentially expressed miRNAs listed in Table 1 (FDR = 0.1), obtained with expression data from non-proliferating Rasless MEFs (lanes 17–24); proliferating control K-Ras^lox (H-Ras^-/-; N-Ras^-/-) cells expressing only K-Ras and the same cells transfected with the empty vectors (lanes 1–8), or BRAF-rescued (lanes 9–12) and MEK1-rescued MEFs (lanes 13–16). The intensity of color saturation in each miRNA box (ranging from 3 to 14 on a log₂ scale) provides a quantitative estimation of its expression level. Red: overexpression. Green: repression. Black:unchanged expression signals relative to controls. Lanes 1–4: K-Ras^loxcell lines DU315-6 (lanes 1, 2) and DU244-1 (lanes 3, 4). Lanes 5–8: K-Ras^lox + empty puromoycin resistance vector cell line MCL23-1 (5–8). Lanes 9–16:BRAF-rescued cell line LG7-6 (lanes 9–12) and MEK1-rescued cell line MCL1-6 (lanes 13–16). Lanes 17–24: Rasless cell lines 6-d 4OHT-treated DU315-6 (lanes 21, 22), 12-d 4OHT-treated DU315-6 (lanes 19, 20), 6-d 4OHT-treated DU244-1 (lanes 23, 24) and 12-d 4OHT-treated DU244-1 (lanes 17, 18).