Figure 2.
Identification of pathway modifiers using a cell-based RNAi assay. (A) Diagrammatic of the 6x2xDraf Luc STAT reporter construct containing 12 STAT92E binding sites (shown with essential residues underlined) and pAct5C-RL used as transfection control. (B) Outline of the paracrine assay system. (C) The effect of dsRNA treatment on the paracrine mode of pathway activation. JAK/STAT pathway activity in each case is shown as fold luciferase activity, and mock transfection corresponds to the basal level of pathway activity with full reporter activity defined as 1 (dashed line). dsRNA-targeting Rh5 and lacZ show no effect, stat92E dsRNA treatment results in an almost basal level of reporter activity, and dsRNA treatment knocking down socs36E results in threefold activation. The seven candidate genes identified in this screen show statistically significant reduction in reporter activity: cip4, P = 0.0002; CG4306, P = 0.001; CG8443, P = 0.0007; Mob1, P = 0.001; CtBP, P = 0.05; did, P = 0.015; and Ten-m, P = 0.012.