4E-BP mutant flies are normal in size. (A) Newly hatched L1 larvae of genotypes w¹¹¹⁸ (“control”) and Thor² (“4E-BP null”) were seeded in vials at a density of 50/vial and grown at 25°C. Male adult flies were then aged 3 d and weighed in batches of 40. Average fly weights for control and 4E-BP-null flies were 0.75 and 0.74 mg, respectively. Error bars indicate standard deviations of 0.005 and 0.002 mg/fly, respectively. (B) Quantitative real-time RT–PCR was performed on RNA extracted from indicated tissues of wandering third instar larvae. The values shown are 4E-BP transcript levels normalized to rp49 transcript levels. Error bars show standard deviation on the assay done in triplicate.

4E-BP mutant flies have impaired survival under starvation conditions. Control w¹¹¹⁸ (solid line) and 4E-BP-null flies (dashed line) were raised under controlled conditions, aged 3 d, and starved; 20 males per vial (0.8% agarose/PBS). Error bars show standard deviation on the assay done in triplicate.

4E-BP regulates fat content. (A) w¹¹¹⁸ (“control”) or Thor² (“4E-BP null”) male flies were raised under controlled conditions and aged for 3 d on normal food or normal food supplemented with 50 μM rapamycin. Total body triglycerides and protein were measured, and the ratio of triglycerides to protein is shown. Measurements were done on batches of eight male flies in triplicate. Error bars show standard error. Average values are 5.8 μg triglycerides/μg protein (control flies, normal food), 5.6 μg triglycerides/μg protein (4E-BP-null flies, normal food), 7.5 μg triglycerides/μg protein (control flies on rapamycin), or 6.4 μg triglycerides/μg protein (4E-BP-null flies on rapamycin). (B) Control or 4E-BP-null male flies were raised under controlled conditions and aged for 3 d on normal food or normal food supplemented with 50 μM rapamycin. Flies were then starved on 0.8% agarose/PBS either supplemented with 50 μM rapamycin or not. Batches of 20 males were assayed in triplicate. Error bars show standard deviation. (C) Flies of various genotypes were grown and aged for 3 d under controlled conditions. Total body triglycerides and protein were measured for each, and the ratio of triglycerides to protein is shown. Three batches of eight males were used for each. (D) Immunoblots of S2 cells transfected to express 4E-BP or the double-alanine mutant 4E-BP(AA). Cells were treated with insulin or not as indicated to induce Tor activity. (Lower panel) Probed with antibody to 4E-BP to show relative loading. (Upper panel) Probed with antibody specific to the phosphorylated form of 4E-BP.

Stress induction of 4E-BP expression is FOXO-dependent. Quantitative real-time RT–PCR on RNA from whole adult control w¹¹¹⁸ flies (A) or FOXO^21/25 mutant flies (B). Larvae were grown under controlled conditions. Adult males were aged for 3 d and then placed in vials containing 0.8% agarose in PBS (“starved”) or supplemented with 10% sucrose or 10%sucrose + 5% H₂O₂. The values shown are 4E-BP transcript levels normalized to rp49 transcript levels. Error bars show standard deviation on the assay done in triplicate.

4E-BP mutant flies burn their fat stores more quickly than control flies. (A) Total body triglycerides and total body protein were measured after controlled growth and aging. Plotted values are triglyceride levels normalized to protein levels. Batches of eight males were assayed, and the experiment was done in quadruplicate. Average values were 4.3 and 4.6 μg triglycerides/μg protein for control and 4E-BP-null flies, respectively. Error bars show standard error. (B) Total triglycerides were measured for flies treated as in A. Batches of eight males were measured in duplicate. Values show triglyceride levels as a percentage of prestarvation levels ± standard error. (C,D) Flies of indicated genotypes were grown under controlled conditions and aged for 3 d. Total body triglycerides were measured for half of the flies immediately, and for the other half after 30 h of starvation on PBS/agarose. Three batches of eight males were used for each measurement. Error bars show standard deviation.