Figure 1

RNAi-based screen to identify components of the Imd signaling cascade in S2 cells. (A) RNAi-mediated gene silencing effectively and specifically decreases the mRNA level of the targeted gene in S2 cells. A total of 2.5 10⁶ S2 cells in six-well plates containing 3 ml of medium were incubated with or without 10 g of CG5210 dsRNA for 48 h. The expression level of more than 13 500 genes was measured using Affymetrix Drosophila genechips. Relative expression levels of all genes are shown. Each point represents the average mRNA expression level of three independent CG5210 RNAi treatments compared pairwise to untreated S2 cells. Arrowhead indicates the dot representing the CG5210 mRNA level. (B) dsRNA treatments targeting ribosomal proteins moderately affect the translation rate of Act5C in S2 cells. Expression of 40 genes coding for ribosomal proteins was silenced using RNAi, and translation rate of Act5C--gal was measured in relation to GFP RNAi-treated controls. Gene-specific -galactosidase activities were thereafter compared to z-scores reported by Boutros et al (2004). There was a significant correlation between Act5C--gal activities and z-scores (see also Supplementary Table SI). (C) Relish RNAi blocks the Imd pathway activity in a dose-dependent manner in S2 cells. Att-luc plasmid was used to measure the activity of the Imd pathway and Act5C--gal to normalize the results. A total of 5.0 10⁵ S2 cells in 500 l of medium were treated with the total of 2 g of the indicated dsRNA(s). GFP dsRNA was used as a negative control. Imd pathway was induced with heat-killed E. coli, and luciferase and -galactosidase activities were measured 24 h afterwards. Data are shown as means.d., N3.