Figure 6. RNAi and inducible immune pathways are functional in Hsf-deficient flies.

(a) Eye phenotype of 5 to 7-day-old flies expressing a hairpin RNA targeting the Drosophila Inhibitor of Apoptosis thread (th^RNAi) and Heat shock factor (Hsf  ^RNAi). The genetic background of the Hsf  ^RNAi line was used as a control (Ctrl). No eye phenotype was observed in control and Hsf  ^RNAi flies that do not expressing the th^RNAi transgene. Five representative images are shown for each genotype. (b) In vivo RNAi reporter assay. Reporter plasmids encoding Firefly (Fluc) and Renilla (Ren) luciferase were transfected along with Fluc specific dsRNA or non-specific control dsRNA in Hsf ⁴ and Dcr2^-/- mutant flies and their wild-type controls (CnBw and y¹w¹, respectively). Reporter gene activity was measured in fly lysates at three days after transfection. Fold silencing by Fluc dsRNA relative to control dsRNA was calculated and presented as the percentage of silencing relative to wild-type controls (see Fig. S6 for Fluc/Ren ratios of all samples). Bars represent mean and s.d. of three pools of five flies for each genotype. (c) Expression of inducible immune genes at 24 hpi with DCV (TCID₅₀ = 10,000) determined by RT-qPCR in wild-type or Hsf ⁴ mutant flies. Expression of the gene of interest was normalized to transcript levels of the housekeeping gene Ribosomal Protein 49 and expressed as fold change relative to mock infection (Tris buffer). Data are mean and s.d. of three independent pools of 15 female flies for each genotype. Student’s t-tests were used to compare the difference in expression between wild-type and mutant flies (*P < 0.05).