(A) Upon Rel or Rel/pnt knockdown cells behave normally, growth is inhibited upon pnt knockdown alone. (B) Dsor1 inhibitor treatment attenuated this alleviating interaction. Scale bar = 30 µm. Images are pseudo colored, DNA/DAPI = blue, FITC/α-tubulin = green. (C) Quantified negative treatment-sensitive interaction between Rel and pnt. The trajectory of the MEK inhibitor treatment is lower than the solvent control condition for cell count interaction indicating synthetic lethality under MEK inhibition. (C’) Actin major axis shows a strong positive interaction (cells are enlarged like under pnt knockdown). Error of fit is shown as 95% confidence interval. Dashed lines show trendlines of a treatment wise model fit. (D, E) Expression of candidate and marker genes assessed by qPCR (3 days RNAi treatment, n = 3, log2 foldRLUC, mean ±s.e.m., t-test) on S2 cells. (D) pnt expression is reduced upon pnt and Pvr knockdown. Rel knockdown does not rescue pnt expression. Rel expression is increased upon pnt and Pvr knockdown and decreased upon Rel knockdown. Upon pnt and Rel knock down, Rel expression is rescued to normal levels. (E) Pvf2 expression is induced only upon Rel/pnt double knockdown. This leads to increased expression of sty and RasGAP1. RasGAP1 knockdown increases sty expression and decreases RasGAP1 expression. (D–E) *=p < 0.05, **=p < 0.01. (F) A model summarizes the qPCR results in context of the Ras signaling cascade. Dashed lines are transcriptional interactions, solid lines are protein-protein interactions. All black interactions are known, while the green interaction is inferred from the data. Blue arrows indicate that Pvf2, sty and RasGAP1 were upregulated upon Rel/pnt co-knockdown and by that Ras pathway activity was restored. A similar pattern could be observed upon RasGAP1 knockdown, which causes intrinsic hyper-activation of Ras signaling by constitutive Ras activation (measured by upregulation of sty, red arrows).