Inactivation of Icmt inhibits cell proliferation induced by expression of an endogenous K-RAS^G12D allele in mouse embryonic fibroblasts. (A) Proliferation of immortalized Icmt^fl/+K^LSL and Icmt^fl/flK^LSL cells infected with Cre- and β-gal-adenoviruses. Cre-adenovirus-treatment of Icmt^fl/+K^LSL and Icmt^fl/flK^LSL fibroblasts produced Icmt^Δ/+K^G12D and Icmt^Δ/ΔK^G12D cells that expressed endogenous K-RAS^G12D and that had one or both Icmt alleles inactivated, respectively. Data are mean plus or minus SEM of one cell line assayed in triplicate. Similar results were obtained in 5 independent experiments. (B) Cells from a focus formation assay taken 12 days after plating (original magnification ×10; 0.22 NA objective). (C) Proliferation of primary Icmt^fl/+K^LSL and Icmt^fl/flK^LSL cells infected with β-gal- (2 left bars) and Cre-adenoviruses (2 right bars). A total of 10⁴ cells were plated and counted after 23 days. Data are one cell line for Icmt^fl/+K^LSL and the mean of 2 cell lines for Icmt^fl/flK^LSL. (D) PCR amplification of genomic DNA from cells of a typical experiment in panel A showing the deletion of the Icmt^fl allele and the stop cassette in the promoter of the Kras2^LSL allele and the appearance of the Icmt^Δ band and the activated Kras2^G12D band in Cre-adenovirus-treated cells (lanes 2 and 4). (E) Western blots showing levels of K-RAS, total RAS, and ACTIN in total cellular extracts from cells in panel A. Similar results were obtained with a different pair of cell lines. For panels B, D, and E, the genotypes of cell lines i through iv are shown in panel A.