Supplementary Figure 1: ISC tumour cells differentiate towards the secretory fate.

(a) Prospero (a, red) in midguts expressing GFP (a, green) with esg^ts for 3 days. (b) Pdm-1 (b, red) in midguts expressing GFP (b, green) with esg^ts for 2 days. (c) Delta (c′) and Prospero (Pros, c^′′) in midguts expressing GFP and N^RNAi (c) with esg^ts for 3 days. esg^lowDl^low cells expressed high levels of Prospero (c–c^′′, arrowheads); esg^high Dl^high ISC-like cells expressed lower levels of Prospero (c–c^′′, arrows). (d) Pdm-1 (d′; d^′′, red) in midguts expressing GFP (d; d^′′, green) and N^RNAi with esg^ts for 2 days. (e,f) Prospero (e′, f′, red) in midguts expressing GFP (e, green) and N^RNAi with esg^ts for 5 days or in midguts recovering for 7 days at 18 °C after expression of GFP (f, green) and N^RNAi for 5 days with esg^ts. (g–j) Mean normalized expression (NE) value (RPKM, log₂) from n = 2 independent experiments of ISC (g) or EE cell (i) associated genes determined by mRNA sequencing of midguts expressing GFP (control, red) or GFP and N^RNAi (tumourous, blue) with esg^ts for 3 days. The adjusted fold change in gene expresson in tumorous midguts (blue), normalized to control midguts (red), is indicated. Benjamini–Hochberg adjusted P-value for Dl, spdo, esg, shg, sc, pros, Rab3 and AstA is 7.71e-322; arm, P = 1.054e-50. The mean NE value (RPKM, log₂) from n = 2 independent experiments of ISC (h) and EE (j) associated genes 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⁺ tumor cells (blue), normalized to esg⁺ cells from control midguts, is indicated. NS, not significant. Benjamini–Hochberg adjusted P-values for Dl is 2.53e-05; spdo, 1.81e-05; sc, 3.48e-14; pros, 1.26e-14; Rab3, 2.52e-19 and AstA, 1.01e-09. (k) Principal component analysis of transcriptional profiles of ISC tumors, intestinal stem cells (ISCs), esg⁺ progenitors, SuH⁺ enteroblasts (EB), enterocytes (EC), enteroendocrine cells (EE) and visceral muscle (VM). DNA is in a′, b′, c^′′′, d^′′′; e–f′ (blue). Scale bars in a–a′, b–b′, c–c^′′′ and d–d^′′′, 25 μm; e–f′, 30 μm.