Supplementary Figure 6: Cytokine induction by ISC tumours is not due to changes in luminal bacteria load.

(a,b) in situ hybridization to upd3 mRNA in midguts from flies infected with Pseudomonas entomophila for 2 days (b; b′, red) and control midguts (no infection; a; a′, red). Cells with high upd3 mRNA are indicated with arrows; with lower upd3mRNA, arrowheads (b–b′). (c) PCR for bacterial 16S rDNA from total genomic DNA isolated from midguts of flies fed normal food or antibiotics for 3 days (undiluted genomic DNA, 1:1). Tumour growth did not increase luminal bacterial load. (d) PCR for the crq gene to detect fly genomic DNA input. (e) Mean number of phosphorylated histone H3 Ser10 positive cells per midgut with s.e.m. in flies expressing GFP (control, -antibiotics: n = 20 midguts; + antibiotics:n = 22 midguts) or N^RNAi (-antibiotics:n = 26 midguts; + antibiotics:n = 28 midguts) with esg^ts for 3 days fed normal food or food containing antibiotics. Reducing gut luminal bacteria to negligible levels had no significant effect on tumour growth. Thus Upd2,3 induction after tumour growth was not due to alterations in luminal bacteria but was a response to tumour growth. (f) Mean number of phosphorylated histone H3 Ser10 positive cells per midgut with s.e.m. of flies fed food alone (n = 31 midguts), Pseudomonas entomophila (P. e.) containing food (n = 24 midguts; Mann–Whitney: P < 0.0001), sucrose alone (n = 33 midguts) or P. e. containing sucrose (n = 15 midguts; Mann–Whitney: P < 0.0001) for 1 day before tumour induction by expressing N^RNAi with esg^ts (weak) for 3 days (food alone). (g) Mean number of phosphorylated histone H3 Ser10 positive cells per midgut with s.e.m. in flies expressing GFP and N^RNAi (n = 36 midguts) or GFP, N^RNAi, cyclin E (cycE) and cdc25/string (stg) (n = 29 midguts, Mann–Whitney: P < 0.0001) with esg^ts (weak) for 3 days. In e, midguts pooled from 2 independent experiments; in f,g, from 3 independent experiments. DNA in a′ and b′ (blue). Scale bars in a–a′, 25 μm; b–b′, 35 μm.