Hh signaling regulates ISC proliferation through the JAK–STAT pathway ligand Upd2. (A and C) mRNA levels of JAK–STAT and EGFR pathway ligands in whole midguts of the flies with indicated genotypes grown at 29°C for 7 (A) or 8 d (C) were measured by RT-qPCR. Numbers indicate fold change over control guts. Three independent experiments were performed, and error bars are standard deviations. **, P < 0.01. (B) Relative levels of Upd2 mRNA in midguts expressing Su(H)^ts>Ptc^RNAi or Su(H)^ts>Smo^RNAi at the indicated time points. Numbers indicate fold change over control guts from three independent experiments. Error bars are standard deviations. (D) The expression of 10XStat-dGFP was elevated in Su(H)^ts>CiΔN adult midguts. (E) Midguts expressing Su(H)^ts>GFP, Su(H)^ts> GFP + Ci^ΔN, Su(H)^ts> GFP plus two copies of UAS-Upd2^RNAi, or Su(H)^ts>GFP + Ci^ΔN + Upd2^RNAi (two copies) for 8 d were dissected out and immunostained for GFP (green), PH3 (red), and Hoechst (blue). (F and G) Quantification of PH3⁺ cells in adult midguts of the indicated genotypes grown at 29°C for 8 (G) or 12 d (H). Of note, only one copy of UAS-Upd2^RNAi was coexpressed with UAS-Ptc^RNAi (G). Three independent experiments were performed and 20 guts were examined for each sample. Error bars are standard deviations. **, P < 0.01. (H) Adult flies expressing Su(H)^ts or Su(H)^ts>Upd2 for the indicated time at 29°C were dissected out and immunostained for PH3 (red) and DRAQ5 (blue). (I) Quantification of PH3⁺ cells in midguts expressing Su(H)^ts (Con) or Su(H)^ts>Upd2 for the indicated time. Three independent experiments were performed and 20 guts were examined for each sample. Error bars are standard deviations. **, P < 0.01. Bars, 50 µm.