Fig. 2: Pax provokes ISC differentiation toward the EC cell.

a–f” Adult midguts of EsgGal4 tubGal80^ts UAS-GFP; UAS-flp Act > CD2>Gal4 (Esg^tsF/O)>Ctrl (a–a”, d–d”), Esg^tsF/O>Pax RNAi (b–b”, e–e”) and Esg^tsF/O > HA-Pax (c–c”, f–f”) were induced for 3 days (a–c”) or 5 days (d–f”). Midguts were immunostained with Arm + Pros (red) (a–f”) and DAPI (nuclei, blue). ECs are marked by Pdm1 (red) (d–f”). ISCs/EBs and EEs/ECs are marked by GFP. White arrows at (a–c”) mark Pros⁺ pre-EE/EE cells. White arrows at (d–f”) mark Pdm1⁺ and GFP⁺EC cells. g Quantification of Pros⁺ pre-EE/EE cells in the whole midgut from the indicated genotypes in (a–c”) (n = 11, 12, 11). The counting of Pros⁺ EE cells was conducted across the entire midgut. h Quantification of GFP⁺ and Pdm1⁺ EC cells in the same region of midgut from the indicated genotypes in (d–f”) (n = 11, 11, 11). i–k” Adult midguts of Esg^ts>Ctrl (i–i”), Esg^ts>Pax RNAi (j–j”) and Esg^ts > HA-Pax (k–k”) were immunostained with NICD (red) and DAPI (nuclei, blue). ISCs and EBs/pre-EEs were marked by EsgGal4-driven GFP expression. l–n’ Adult midguts of NRE-lacZ;Esg^ts>Ctrl (l–l’), NRE-lacZ; Esg^ts>Pax RNAi (m–m’) and NRE-lacZ;Esg^ts > HA-Pax (n–n’) were immunostained with β-gal (red), HA (blue) and DAPI (nuclei, gray). EsgGal4-driven GFP marks ISCs and EBs/pre-EEs. o Quantification of lacZ⁺ EB cells in the same region of midgut from the indicated genotypes in (l–n’) (n = 10,10,10). p–q”’ Adult midguts of Esg^ts>Flag-Pax (p–p”’) and Esg^ts>Flag-Pax; Notch RNAi (q–q”’) were immunostained with Pdm1 (red), Flag (blue), and DAPI (nuclei, gray). EsgGal4-driven GFP marks ISCs and EBs/pre-EEs. r Quantification of the percentage of Pdm1⁺ EC cells in the same region of midgut from the indicated genotypes in (p–q”’) and (Supplementary Fig. 6a–b”) (n = 15,12,11,11). s Total RNA for real-time PCR was collected from whole midguts of the indicated genotypes: Esg^ts>Ctrl and Esg^ts > HA-Pax. Midguts were analyzed 5 days post-induction (n = 3,3). The relative mRNA level was normalized with that of Esg-GFP. t Total RNAs for real-time PCR were collected from whole midguts of the indicated genotypes: Esg^ts>Ctrl and Esg^ts > HA-Pax. Midguts were analyzed 5 days post-induction (n = 3,3). The relative mRNA level was normalized with that of Esg-GFP. u Total RNA for real-time PCR was collected from whole midguts of the indicated genotypes: Esg^ts>Ctrl and Esg^ts>Pax RNAi. Midguts were analyzed 5 days post-induction (n = 3,3). The relative mRNA level was normalized with that of Esg-GFP. Three independent experiments were performed, and the error bars are mean ± SEM. In each box plot, the center line indicates the median, the edges of the box represent the first and third quartiles, and the whiskers extend to the minimum and maximum values. P values of significance (indicated with asterisks, NS no significance P ≥ 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****p < 0.0001) were calculated by two-tailed Student’s t-test (s–u) and one-way ANOVA with Tukey’s test (g, h, o, r). Scale bars: 30 µm. Confocal images were taken from the basal layer of the posterior midgut.