Nef induces apoptosis by activating JNK signaling pathway and inhibits NF-B-dependent immune responses in Drosophila

JCS02312 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. (A) Determination of the expression levels of nef transcript using RT-PCR. All flies were subjected to 2 hours of heat shock to induce nef expression. Ribosomal protein 49 (Rp49) was used as an internal control. RT products were amplified 25 times to detect nef and Rp49 transcripts in PCR reactions. (B-G) Enhanced puc expression caused by Nef-dependent JNK activation. Wing imaginal discs were stained with X-gal to determine b-galactosidase expression. (B) MS1096/X;; puc-lacZ/+. (C) MS1096/X;; UAS-nef/puc-lacZ. (D) MS1096/X; UAS-hep/+; puc-lacZ/+. (E) en-GAL4/+; puc-lacZ/+. (F) en-GAL4/+; UAS-nef/puc-lacZ. (G) hep^r75/Y; en-GAL4/+; UAS-nef/puc-lacZ. MS1096-GAL4 and en-GAL4 drive GAL4 expression in the whole region and the posterior region of Drosophila wing, respectively.

• Supplemental Figure 2 -

  Fig. S2. TUNEL-positive apoptosis in nef-expressing clones. Apoptotic cells and nef-expressing clones in wing imaginal discs were determined by TUNEL assays (red) (A,D) and GFP signals (green) (B,E), respectively. Merged images are shown (C,F). In this experiment, nef-expressing cells were marked by nuclear GFP signals. Pictures were taken under a magnification of 4003 (A-C) 40003 (D-F).

• Supplemental Figure 3 -

  Fig. S3. JNK-independent inhibition of melanization by Nef. (A-D) Inhibition of melanization by Nef. Comparison between control and the nef-expressing larvae (hsp70>nef or nef) at 10 minutes (A) or 1 hour (A-C) after bacterial infection. (D) The effect of Nef on melanization was measured in combination with reduced hep gene dosage or simultaneous expression of JNK^DN. Phenotype evaluation: +, spot-like melanization at the infected region; ++, localized melanization around the infected region, affecting less than one-quarter of the whole body; +++, systemic melanization, affecting more than one-quarter of the whole body. nef expression was induced by 2 hours of heat shock at 37°C before bacterial infection. Pictures were taken under a magnification of 12.53 (A) or 6.53 (B,C). (E) Induction of Dpt mRNA by bacterial infection was determined by northern-blot analysis (top). The expression level of puc transcript was also measured to confirm JNK activation by hep^CA expression (middle). 18S rRNA was used as a loading control (bottom). To induce hep^CA expression, larvae were heat shocked for 30 minutes at 37°C and subsequently incubated for 90 minutes at 25°C, then collected before (uninfected) or after (infected) bacterial infection. Fly genotypes used were as follows. C: Control, hsp70-GAL4/+. hep^CA: hsp70-GAL4/+; UAS-hep^CA/+.