Supplementary Figure 5: Cytokines are induced by ISC tumour growth and not by enteroendocrine cell expansion.

(a) Mean fold change (log₂) with s.e.m. from n = 4 independent experiments in Delta and cytokine (upd1, upd2, upd3) mRNA levels determined by quantitative PCR on midguts bearing 7 day old neur^IF65/− clones. Fold changes were normalized to control midguts bearing 7 day old wild-type clones. (b) The mean normalized expression (NE) value (RPKM, log2) from n = 2 independent experiments of cytokines (upd1-3) and JAK-STAT target Socs36E determined by mRNA sequencing of esg⁺ cells sorted from control midguts expressing GFP (red) or from tumorous midguts expressing GFP and N^RNAi (blue) with esg^ts for 3 days. The adjusted fold change in gene expression in sorted esg⁺ tumour cells (blue), normalized to esg⁺ cells from control midguts, is indicated. NS, not significant. The Benjamini–Hochberg adjusted P-value for upd2 is 1.36e-10. (c) β-galactosidase (c–c′, red) in midguts of flies carrying upd-lacZ and expressing N^RNAi with esg^ts for 2 days. (d) Mean fold change (log₂) with s.e.m. of cytokine (upd 1-3) and EGF ligand (spi, krn, vn) mRNA levels determined by quantitative PCR on midguts expressing N^RNAi for 2 days (n = 3 independent experiments), sc for 7 days (n = 3 independent experiments), and SuH^RNAi for 5 days (n = 4 independent experiments) with esg^ts. Fold changes were normalized to control whole midguts overexpressing GFP with esg^ts for 2 (for N^RNAi), 7 (for sc) and 5 days (for SuH^RNAi). ND, not determined; NS, not significant. Overexpression of the pro-secretory factor Scute (Sc) expanded the number of Pros⁺ EEs (Ast⁺). (e,f) Phospho-histone H3 Ser 10 (red) in control (e) or upd2-3 mutant (Δ2-3) (f) midguts expressing GFP (green) and N^RNAi for 14 days with esg^ts (weak). (g) Delta (g; g′, red) and Prospero (g^′′) in midguts expressing GFP (g′, green) and N^RNAi with esg^ts for 4 days. (h) β-galactosidase (red) and D–GFP (green) in midguts of flies carrying Dl-lacZ and 10X STAT-DGFP. (i,j) D–GFP (i, j; i′, j′, green) in midguts of flies carrying 10X STAT-DGFP and expressing N^RNAi (i) or N^RNAi and dome^ΔCYT (j) with esg^ts for 4 days. Rare, large ISC tumour shown in j. DNA in c′, e, f, h, i′, and j′ (blue). Scale bars in c–c′, 35 μm; e, 25 μm; f, 30 μm; g–g^′′, 20 μm; h, 4 μm; i–j′, 30 μm.