Figure 3. Components of the RAS pathway are required for MSB2 expression and FG pathway regulation.

(A) Expression of FG pathway-dependent reporters in wild-type cells and the ras2Δ mutant. Cells containing plasmid born lacZ fusions [65] were grown to mid-log phase in SD-LEU medium to maintain selection for the plasmids and harvested by centrifugation. β-galactosidase assays were performed in duplicate. Y-axis, β-galactosidase activity expressed in Miller Units (U). Error bars represent the standard deviation between experiments. (B) Invasive growth assays. For the plate-washing assay at left, equal concentrations of cells were spotted onto YEP-GAL medium and incubated for 2d. The plate was photographed (YEP-GAL) and washed in a stream of water to reveal invaded cells (Washed). For the single-cell invasive growth assay at right, cells were grown on S+GAL medium at low density for 16 hours and photographed at 100X. Bar, 10 microns. (C) MSB2^AG-lacZ expression in wild type, ras2Δ, sdc25Δ, flo8Δ, ste12Δ, and ras2Δ pde2Δ mutants grown in SD or S+GAL medium. Y-axis, β-galactosidase activity expressed in Miller Units (U). The experiment was performed in duplicate, and error bars represent standard deviation between experiments. (D) Expression profiling of cells containing an activated FG pathway (either containing an activated allele MSB2^Δ100–818 or expressing GAL-MSB2) in wild-type cells the ras2Δ and ste12Δ mutants. Heat map of DNA microarray comparisons is shown. Red, fold induction; green, fold repression; black, no change; grey, N/D. Each spot represents the average of 3 independent comparisons. Genes involved in nutritional (N) scavenging or stress response (S) are marked in yellow (column N/S). Targets of the RIM101 pathway are shown in purple [19]. The complete microarray dataset, including the raw data, statistical analysis, and functional classification of genes is presented in Table S6.