(A–F) Transverse sections (200 μm) through the intestinal bulb region of untreated WT (A) and ttis450 (B) larvae at 120 hpf or larvae previously treated for 14 h with rapamycin and/or chloroquine (C–F) stained with rhodamine phalloidin to detect F-actin (red), Hoechst 33342 to detect DNA (blue) and the LC3B antibody to detect LC3II–containing autophagosomes (green puncta). (G) The numbers of autophagosomes are increased in chloroquine-treated WT and ttis450 larvae compared to the corresponding untreated larvae. Chloroquine-treated ttis450 larvae contain significantly more puncta than chloroquine-treated WT larvae and similar numbers to WT larvae treated with rapamycin and chloroquine. Rapamycin and chloroquine-treated ttis450 larvae contain significantly more puncta per IEC than the IECs in chloroquine-treated ttis450 larvae and chloroquine and rapamycin-treated WT larvae. Puncta were counted in 20 cells from 3 independent sections using Metamorph. (H) Representative Western blot analysis of whole cell lysates of WT and ttis450 larvae (96 hpf) previously treated for 14 h with rapamycin (10 μM) and/or chloroquine (2.5 μM) using antibodies to LC3B and Actin (loading control). (I) Graphical representation of the data shown in H and two independent analyses. The LC3II signals were quantitated by densitometry. ttis450 larvae treated with chloroquine contain more LC3II than their chloroquine-treated WT siblings and comparable levels to WT larvae treated with rapamycin and chloroquine. Data are represented as mean +/ SD, *p<0.05.