Figure 4: Effects of altered expression of Pvf/Pvr–ERK signalling related genes on cell spreading and Metchnikowin expression.

(a–c) Effect of dsRNA targeting of Erk (a), Pvr (b), or Pvf2 (c) on spreading of Drosophila S2 cells stimulated by 50 nM GBP for the indicated times. Each value represents the mean±s.d. for 3–7 independent determinations. *P<0.05; **P<0.01: Significant differences from control (0 min, EGFP-RNAi) value are indicated by Tukey’s HSD. (d) Effect of dsRNA targeting of Pvr on GBP-induced spreading of plasmatocytes of Drosophila larvae. Plasmatocytes prepared from test larvae were incubated with 20 nM GBP (see Materials and Methods). Average rates of spread plasmatocytes prepared from any test larvae were less than 20% when they were incubated with 20 nM BSA for 30 min. Each value represents the mean±s.d. for six independent determinations. *P<0.05; **P<0.01: Significant differences from control (0 min, Actin-Gal4) value are indicated by Tukey’s HSD. (e) Analysis of hemocyte Mtk expression with Pvr knockdown backgrounds under forced expression of GBP in Drosophila larvae by quantitative real-time PCR. The hs-Gal4-dependent knockdown of Pvr and overexpression of GBP were induced by heat treatment at 35 °C for 30 min twice with 12 h-interval. Infection was induced by stabbing third instar larvae with a thin tungsten needle previously dipped into a concentrated culture of Serratia marcescens 3 h before measuring Mtk expression. Induction of Mtk expression by forced GBP expression was measured in larvae whose Pvr expression was significantly repressed as shown in Supplementary Fig. 8. Each value represents the mean±s.d. for five independent determinations. **P<0.01: Significant differences from control (UAS-dsPvr;UAS-GBP) value are indicated by Tukey’s HSD.