Immunostaining

For lymph gland staining, lymph glands were dissected in ice-cold PBS. After fixation for 30 min in 4% paraformaldehyde, dissected tissues were incubated in blocking solution (PBS containing 0.1% Tween-20% and 5% goat serum) for 30 min and then incubated with primary antibodies diluted in blocking solution at 4°C overnight. After several rinses in PBST, lymph glands were sequentially incubated with secondary antibodies for 2 hr and 4'6-diamidino-2-phenylindole (DAPI) for 10 min before being mounted with Slowfade mounting reagent (Thermo Fisher). For the immunostaining of hemocytes, 10 larvae were bled in 10 μl of PBS and then transferred to an adhesive glass slide. After incubation for 30 min in a humidified chamber, hemocytes were fixed using 4% paraformaldehyde for 10 min, blocked in blocking buffer, and sequentially incubated with primary antibodies, secondary antibodies, and DAPI before being mounted. All samples were observed under a Zeiss Axioskop 2 Plus microscope or a Zeiss LSM510 confocal microscope. The following primary antibodies were used in the study: rabbit anti-PH3 (Millipore, RRID:AB_1977177), mouse anti-P1 and mouse anti-L1 (gifts from Istvan Andó); rabbit anti-ProPO1 (a gift from Erjun Ling); rabbit anti-Dif (a gift from Dominique Ferrandon); mouse anti-Antp (RRID:AB_528082), mouse anti-Dorsal (RRID:AB_528204), mouse anti-Hrs (RRID:AB_2618261) and mouse anti-Mmp1 (RRID:AB_579782) (Developmental Studies Hybridoma Bank); rabbit anti-Rab5 (RRID:AB_882240) and rabbit anti-GABARAP (anti-Atg8) (Abcam, RRID:AB_10861928); mouse anti-Rab11 (BD Biosciences, RRID:AB_397983); rabbit anti-HA (Sigma, RRID:AB_260070); rabbit anti-dFOXO (a gift from Pierre Léopold); rabbit anti-p-Erk (Cell Signaling Technology, RRID:AB_2315112); mouse anti-p-Jun (Santa Cruz, RRID:AB_629275), mouse anti-β-Gal (RRID:AB_430877), and mouse anti-p-JNK (RRID:AB_430864) (Promega). All Alexa Fluor 488- and Alexa Fluor 568-conjugated secondary antibodies were purchased from Thermo Fisher.