Fig. 5: The activation of JNK signaling in ISCs is restricted by N-glycosylation.

a, c–e, g–i Different genetic manipulations are indicated in individual panels. Midguts were stained with anti-pH3 antibodies, and ISC mitoses were quantified by counting pH3⁺ cells. Quantification data represent the mean ± SD (two-tailed unpaired t-test, ^nsP > 0.05, **P = 0.0012 (a), ***P = 0.0005 (a), ***P = 0.001 (h), ****P < 0.0001). N values in individual panels indicate the number of midguts examined. b, k w1118 (control), UAS-Alg3^RNAi, UAS-Alg9^RNAi, and UASp-Pngl were overexpressed by esg^ts. Flies were raised at 18 °C and then shifted to 29 °C for 4 days (b) or 6 days (k) before dissection. Puc-lacZ^E69 was used to indicate the activation of JNK signaling. Nuclei were labeled in blue. f Flies were raised at 18 °C. Two-day-old esg^ts; UAS-GFP female files were shifted from 18 to 29 °C for 2 days and then treated with 5% sucrose (control), Ecc15, or P.e. for 18 h. RT-qPCR was performed on cDNA samples extracted from the FACS-sorted GFP⁺ progenitor cells. Quantification data represent the mean ± SD (n = 3 replicates/treatment/gene, two-tailed unpaired t-test, *P = 0.0227, **P = 0.0077, ***P = 0.0003, ****p < 0.0001). j UAS-grnd^WT-V5 (wild-type Grnd) or UAS-grnd^N63A-V5 (N-glycosylation site mutation form Grnd) was overexpressed in progenitors using esg^ts at 29 °C for 7 days. esg^ts > w1118 flies were used as controls. Five midguts per sample were dissected for western blot analysis using anti-V5/-tubulin antibodies for blotting. l The data presented in (a–k) collectively support a model in which ALG3/9-mediated N-glycosylation of Grnd functions as a pivotal switch for JNK activation in progenitor cells upon infection. Images in (b) are representative of three independent experiments. Images in (j, k) are representative of two independent experiments. Scale bars: 15 μm. Source data are provided as a Source Data file.