Activation of insulin and wingless signaling pathways in Drosophila S2 cells. (a) Schematic representation of the insulin and wingless signal transduction cascades and controls of their activation in our experimental system. Key protein components and their interactions for each pathway are schematized. Dashed lines represent cell and nuclear membranes. C and N indicate cytoplasm and nucleus, respectively. Stimulation of insulin signaling from 0-8 h was monitored by western blotting using an anti-phospho-Akt antibody (left panel). Activation of the wingless pathway, achieved through RNA interference (RNAi)-mediated depletion of axin (axn), resulted in the nuclear accumulation of Armadillo (Arm) as assessed by western blot analysis and activation of a known target gene, naked cuticle (nkd) monitored by RT-PCR (right lower panel). Amplification of tubulin (tub) transcripts served as loading control. The arrow indicates the time-point used for our microarray analysis. (b) Distribution of genes showing transcriptional up- and down-regulation upon activation of insulin and wingless. (c) Validation of microarray predictions by quantitative RT-PCR. Three genes are shown for each pathway. Results are presented as log2 ratio of signals obtained under conditions of pathway activation and controls. Z-scores predicted by microarray data analysis are indicated below the graphs.